Methods of Treating Sickle Cell Disease and Related Disorders Using Fumaric Acid Esters

ABSTRACT

Methods of using one or more fumaric acid esters or pharmacologically active salts, derivatives, analogues, or prodrugs thereof to increase expression of fetal hemoglobin (HbF) are disclosed. The methods typically include administering to a subject an effective amount of one or more fumaric acid esters optionally in combination or alternation with hydroxyurea to induce HbF expression in the subject in an effective amount to reduce one or more symptoms of a sickle cell disorder, a hemoglobinopathy, or a beta-thalassemia, or to compensate for a genetic mutation is the human beta-globin gene (HBB) or an expression control sequence thereof. Pharmaceutical dosage units and dosage regimes for use in the disclosed methods are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of co-pending U.S. application Ser. No.14/105,893, entitled “Methods of Treating Sickle Cell Disease andRelated Disorders Using Fumaric Acid Esters” by Vadivel Ganapathy andPamela M. Martin, filed Dec. 13, 2013, which claims the benefit of andpriority to U.S. Provisional Application No. 61/737,360 filed Dec. 14,2012, all of which are herein incorporated in their entirety byreference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Agreement NEIEY018053 awarded the National Institutes of Health. The government hascertain rights in the invention.

FIELD OF THE INVENTION

The field of the invention is generally related to compositionsincluding fumaric acid esters and methods of their use for HbF (γ-globingene) induction.

BACKGROUND OF THE INVENTION

Sickle-cell disease (SCD), also known as sickle-cell anemia (SCA) anddrepanocytosis, is an autosomal recessive genetic blood disorder causedby a point mutation in the β-globin chain of hemoglobin. SCD ischaracterized by red blood cells that adopt an abnormal, rigid, sickleshape, referred to as “sickling” under low-oxygen conditions. Repeatedepisodes of sickling can damage the blood cell's membrane and decreaseits elasticity. Sickled cells can fail to return to normal shape whennormal oxygen tension is restored. As a consequence, these rigid bloodcells are unable to deform as they pass through narrow capillaries,leading to vessel occlusion and ischemia. The actual anemia of theillness is caused by hemolysis, the destruction of the red cells, causedby their misshapes.

Normally, humans have Hemoglobin A, which consists of two alpha and twobeta chains, Hemoglobin A2, which consists of two alpha and two deltachains and Hemoglobin F, consisting of two alpha and two gamma chains intheir bodies. Of these, Hemoglobin A makes up around 96-97% of thenormal hemoglobin in humans. Fetal hemoglobin (also hemoglobin F or HbF)is the main oxygen transport protein in the fetus during the last sevenmonths of development in the uterus and in the newborn until roughly sixmonths old. Functionally, fetal hemoglobin differs most from adulthemoglobin in that it is able to bind oxygen with greater affinity thanthe adult form, giving the developing fetus better access to oxygen fromthe mother's bloodstream.

In newborns, fetal hemoglobin is nearly completely replaced by adulthemoglobin by approximately six months postnatally. However, HbF can bereactivated pharmacologically, an approach that has been investigated asa treatment for symptoms and complications of SCD.

Several classes of pharmacological agents that reactivate γ-globin genetranscription, thereby inducing HbF production, have been identified.However, the S-stage cytotoxic drug hydroxyurea (HU) is the first and atpresent only FDA-approved drug for treatment of SCD. While HU has beenshown to reduce vaso-occlusive episodes and associated complicationssuch as pain and acute chest episodes in a large number of sickle-cellpatients treated, there are a number of limitations to using HU such asbone marrow suppression, concerns over long-term carcinogeniccomplications, and a 30% non-response rate.

Therefore, it is an object of the invention to provide compositions andmethods for treating subjects with one or more mutations in thebeta-globin gene (HBB), or an expression control sequence thereof.

It is another object of the invention to provide compositions andmethods for treating subjects with sickle cell disease, betathalassemia, or variants or related diseases or conditions thereof.

It is another object of the invention to provide compositions andmethods for reducing one or more symptoms of sickle cell disease, betathalassemia, or variants or related diseases or conditions thereof.

It is a further object of the invention to provide treatments for sicklecell disease with fewer, or less severe side effects, greater efficacy,greater response rate, or combinations thereof compared to existingtherapies such as hydroxyurea.

SUMMARY OF THE INVENTION

Monomethylfumarate induces γ-globin expression and fetal hemoglobinproduction in human erythroid and retinal pigment epithelial cells.Therefore, methods of treating sickle cell disease (SCD) orcomplications of SCD include administering an effective amount of onemore fumaric acid esters or pharmacologically active salts, derivatives,analogues, or prodrugs thereof to induce or increase expression of fetalhemoglobin (HbF) in a subject in need thereof are disclosed. Anothermethod for treating SCD or complications related to SCD includesadministering one or more fumaric acid esters in combination oralternation with hydroxyurea (HU). In one aspect, the subject treatedwith the combination of fumaric acid ester and HU is typicallyunresponsive or does not respond well to HU treatment alone. Preferredsubjects for treatment with the combination of fumaric acid esters andHU have reduced expression of OCTN1 relative to subjects that respondwell to HU treatment alone.

Methods for treating retinopathy due to SCD includes administering oneor more fumaric acid esters optionally in combination with HU in anamount effective to increase HbF in retinal pigment epithelial cells

Examples of suitable fumaric acid esters include, but are not limited tomonoethyl fumarate (MEF), monomethyl fumarate (MMF), diethyl fumarate(DEF), and dimethyl fumarate (DMF). In a preferred embodiment, thefumaric acid ester is MMF, DMF, or a combination thereof.

The one or more fumaric acid esters or pharmacologically active salts,derivatives, analogues, or prodrugs thereof are administered to asubject in an effective amount to increase HbF in the subject.

The one or more fumaric acid esters or pharmacologically active salts,derivatives, analogues, or prodrugs thereof can also be administered inan effective amount to increase HbF expression in a subject in needthereof to reduce one or more symptoms of a sickle cell disorder in thesubject. The sickle cell disorder can be a sickle cell disease such assickle cell anemia. Typically, the subject has at least one allele ofsickle cell hemoglobin (HbS). In some embodiments, the subject has oneallele of HbS and one allele of hemoglobin C (HbC), one allele ofhemoglobin E (HbE), one allele of β-0 thalassemia, or one allele ofβ+thalassemia. In some embodiments, the subject has two alleles of HbS.

The fumaric acid esters or pharmacologically active salts, derivatives,analogues, or prodrugs thereof can be used in combination or alternationwith another therapeutic agent to treat SCD or complications of SCD. Forexample the fumaric acid esters can be combined with HU. The combinationof fumaric acid esters with HU can be formulated in a unit dose form.Thus, one embodiment is a pharmaceutical composition comprising afumaric acid ester and HU, optionally including an excipient. Anexemplary complication of SCD that can be treated with the disclosedcompositions includes but is not limited to retinal complications.

The one or more fumaric acid esters or pharmacologically active salts,derivatives, analogues, or prodrugs thereof can be administered in aneffective amount to increase HbF expression in a subject in need thereofto reduce one or more symptoms of a beta-thalassemia in the subject. Thebeta-thalassemia can be, for example, thalassemia minor, thalassemiaintermedia, and thalassemia major.

In some embodiments, the one or more fumaric acid esters orpharmacologically active salts, derivatives, analogues, or prodrugsthereof is administered to a subject in an effective amount to increaseHbF expression in the subject in need thereof to compensate for amutation in the human beta-globin gene. Compensating for a mutation inthe human beta globin gene includes inducing expression of HbF.

Methods of increasing HbF expression in hemoglobin synthesizing cellsare also disclosed. The methods typically include contacting cells withan effective amount of a fumaric acid ester, or pharmacologically activesalt, derivative, analogue, or prodrug thereof to increase HbFexpression in the cells. The contacting can occur in vitro or in vivo.In some embodiments the cells are erythroid precursor cells.Alternatively, the cells are non-erythroid cells such as macrophage,retinal pigment cells, or alveolar epithelial cells.

The one or more fumaric acid esters or pharmacologically active salts,derivatives, analogues, or prodrugs thereof can be in a pharmaceuticalcomposition. The dosage can be between 1 mg/kg to about 50 mg/kg. Thedosage can be between 0.1 g and 2.0 g per day. The fumaric acid ester,or pharmacologically active salt, derivative, analogue, or prodrugthereof can be administered as part of a dosage regime. The dosageregime can include dose escalation.

The current labeled dosing of hydroxyurea for sickle cell disease callsfor the administration of an initial dose of 15 mg/kg/day in the form ofa single dose, with monitoring of the patient's blood count every 2weeks. If the blood counts are in an acceptable range, the dose may beincreased by 5 mg/kg/day every 12 weeks until the MTD of 35 mg/kg/day isreached. Pharmaceutical compositions can contain 1 mg/kg to 50 mg/kg offumaric acid ester, preferably MMF, in combination with 1 mg/kg to 35mg/kg of HU.

For example, a dosage regime for treatment of a sickle cell disorder caninclude administering to a subject with a sickle cell disorder a lowdose of a fumaric acid ester, or pharmacologically active salt,derivative, analogue, or prodrug thereof and administering to thesubject escalating doses of the fumaric acid ester, or pharmacologicallyactive salt, derivative, analogue, or prodrug thereof until the dose iseffective to reduce one or more symptoms of the sickle cell disorder.

Some of the disclosed methods include administering to the subject asecond active agent, for example, vitamin supplements, nutritionalsupplements, anti-anxiety medication, anti-depression medication,anti-coagulants, clotting factors, anti-inflammatories, steroids such ascorticosteroids, analgesic, etc. In some embodiments, the compositionsare co-administered in combination with one or more additional activeagents for treatment of sickle cell disease, beta-thalassemia, or arelated disorder. Such additional active agents may include, but are notlimited to, folic acid, penicillin or another antibiotics, preferably aquinolone or macrolide, antivirals, anti-malarial prophylactics, andanalgesics to control pain crises. In some embodiments, the compositionsare co-administered with one or more additional agents that increaseexpression of HbF, for example, hydroxyurea.

Methods of selecting a subject with a mutation in a beta-globin gene fortreatment are also disclosed. The methods typically include genotypingthe beta-globin gene and expression control sequence thereof in DNAisolated from a biological sample obtained from the subject; determiningif the beta-globin gene or expression control sequence includes amutation; selecting the subject for treatment if the beta-globin gene orexpression control sequence includes a mutation; and treating thesubject with an effective amount of one or more fumaric acid esters, orpharmacologically active salts, derivatives, analogues, or prodrugsthereof.

Still another method of treatment provides administering fumaric acidesters in combination or alternation with HU to SCD subjects that areunresponsive to HU treatment alone. For example, methylmonofumaric acidester can be administered to enhance the update of HU in subjects thatare typically unresponsive to HU. Unresponsive to HU treatment meansthat the subject having SCD does not expiring a significant therapeuticeffect for treating their SCD from HU treatment. The increase in uptakeof HU can also be accompanied by an increase in HfB expression.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing the change in γ-globin mRNA expression(fold change compared to untreated control (“UT”)) in KU812 cells overtime (hours) following treatment with 100 μM monomethylfumarate (MMF)treatment. Data are represented as means±standard error of the mean(SEM); *P<0.05, **P<0.01 and ***P<0.001.

FIG. 2 is a bar graph showing the change in γ/β-globin mRNA expression(fold change compared to untreated control (“UT”)) in KU812 cells overtime (hours) following treatment with known fetal hemoglobin (HbF)inducers (10 mM Cysteine (Cys), 50 μM Hemin, 2 mM sodium buytrate NaB,2000 nM suberoylanilide hydroxamic acid (SAHA), 105 nM cyclic peptideFK228 (depsipeptide), 100 μM hydroxyurea (HU)). Reproduced from Makala,et al., Anemia. 2012; 2012: 428137. Epub 2012 May 14.

FIG. 3 is a bar graph showing the ratio of luciferase gene expression(γ) to γ+2× renilla gene expression (γ+2β) (fold change compared tountreated control (“UT”)) in KU812 cells over time (hours) treated withincreasing doses of monomethylfumarate (MMF). The assayed KU812 cellsexpress a μLCRβprRlucγprFluc construct containing a 3.1-kb μLCR cassettelinked to a 315-bp human β-globin promoter driving the renilla and a1.4-kb Aγ-globin promoter driving the firefly luciferase genes.

FIG. 4 is a bar graph showing the ratio of luciferase gene expression(γ) to γ+2× renilla gene expression (γ+2β) (fold change compared tountreated control (“UT”)) in KU812 cells over time (hours) treated withincreasing doses of dimethylfumarate (DMF). The assayed KU812 cellsexpress a μLCRβprRlucγprFluc construct containing a 3.1-kb μLCR cassettelinked to a 315-bp human β-globin promoter driving the renilla and a1.4-kb Aγ-globin promoter driving the firefly luciferase genes.

FIG. 5A is a photograph of a gel showing γ-globin expression in humanRPE cells (ARPE-19 cell line) analyzed by reversetranscriptase-polymerase chain reaction (RT-PCR) at various time pointsafter treatment with 100 μM monomethylfumarate (MMF). FIG. 5B is a bargraph showing γ-globin expression in human RPE cells (ARPE-19 cell line)analyzed by real-time quantitative PCR (qPCR) at various time pointsafter treatment with 100 μM monomethylfumarate (MMF). Data arerepresented as mean±SEM; *P<0.05, **P<0.01.

FIG. 6 is a bar graph showing γ-globin mRNA expression in primary RPEcells (fold change) isolated from the eyes of humanized mice analyzed byreal-time quantitative PCR (qPCR) following treatment withmonomethylfumarate (MMF) at various concentrations ranging from 0-1000μM for a period of 9 hours. Data are represented as mean±SEM; *P<0.01,**P<0.001.

FIGS. 7A-7E show the induction of γ-globin gene expression and HbFproduction by dimethylfumarate (DMF) and monomethylfumarate (MMF) inerythroid cells. The dual luciferase reporter KU812 stable cell line(1×10⁶ cells/assay) was treated with varying concentrations (0-1000 μM)of DMF 7A or MMF 7B for 48 h. Cells cultured in the absence of the drugswere included as controls (UT, untreated). Firefly luciferase andrenilla luciferase activity was measured for γ-globin and β-globinpromoter activity, respectively. Trypan blue exclusion was used tomonitor cell viability. FIG. 7A is a bar graph of γ/γ+β (Fold Change)versus DMF (μM). FIG. 7B is a bar graph of γ/γ+β (Fold Change) versusDMF (μM). FIG. 7C is a bar graph of γ/β mRNA level of primary humanerythroid progenitors grown in liquid culture treated with DMF, MMF, orHU (μM). The level of γ-globin and β-globin expression was normalized toGAPDH before the γ/β mRNA ratio was calculated. FIGS. 7D(1)-D(4) areline graphs of Relative cell counts versus log fluorescence values foruntreated cell (FIG. 7D(1), cells treated with 100 μM HU (FIG. 7D(2),cells treated with 200 μM DMF (Figure D(3), or cells treated with 1000μM MMF (FIG. 7D(4)). FIG. 7E is a bar graph of FITC Positive Cells (%)in untreated cells (UT, solid bar), cells treated with 200 μM DMF, cellstreated with 1000 μM MMF, and cells treated with 100 μM HU. Data wereexpressed also as the mean concentration of HbF per cell measure.

FIGS. 8A-8F are bar graphs showing globin gene expression and HbFproduction in human RPE cells. FIG. 8A shows qPCR analysis of endogenousα-, β- and γ-globin gene expression (Relative Expression) relative tothat of hypoxanthine-guanine phosphoribosyltransferase 1 (internalcontrol) in the human RPE cell line ARPE-19. FIG. 8B is a bar graphshowing qPCR used to evaluate γ-globin mRNA expression (Fold Change) incontrol (UT, untreated) and monomethylfumarate (MMF)-treated cells (1000μM; 6-24 h). FIG. 8C is a bar graph showing qPCR analysis of endogenousα-, β- and γ-globin gene expression (Relative Globin mRNA/HPRT) relativeto that of hypoxanthine-guanine phosphoribosyltransferase 1 (internalcontrol) in AA (solid bars) and SS (open bars) primary RPE cells. FIG.8D is a bar graph showing qPCR analysis of γ-globin mRNA expression((Fold Change) in control (UT, untreated), MMF-treated (1000 μM) orHU-treated (100 μM; positive control) AA (solid bars) and SS (open bars)primary RPE cells. FIG. 8E is a bar graph of induction of HbF proteinexpression (FITC Positive Cells (%)) by the indicated agents evaluatedin AA (solid bars) and SS (open bars) primary RPE cells by FACS usingthe FITC-conjugated anti-γ-globin antibody and, the number ofFITC-positive cells normalized to isotype controls expressed ingraphical format. HbF protein expression was confirmed by Western blot.MMF (1 mM final concentration) or phosphate buffered saline (PBS; 0.01 MpH 7.4) was injected intravitreally into the eyes of live AA and SS mice(n=6); 24 h later, γ-globin mRNA expression in RPE/eyecup and HbFprotein in intact retina was evaluated by qPCR (FIG. 8F). FIG. 8G is abar graph of γGlobin mRNA Expression (Fold Change) in AA and SS cellstreated with 0.01 M PBS pH7.4 (solid bars) or 1 mM final concentrationof MMF (open bars).

FIG. 9 is a bar graph showing FACS analysis of HbF protein expression.The graph shows Mean Fluorescence Intensity of primary human erythroidprogenitor cells treated with 200 μM DMF, 1000 μM MMF, or 100 μM HU,(UT, untreated). ***p<0.001 compared to untreated control.

FIG. 10 is a bar graph showing β-Globin expression in AA and SS primaryRPE. The expression of human β-globin mRNA (Fold Change) relative tothat of hypoxanthine guanine phosphoribosyl transferase 1 (internalcontrol) was analyzed by qPCR in AA (solid bar) and SS (open bar)primary RPE cells treated (24 h) with MMF (1000 μM) or HU (100 μM); UT,untreated control. *p<0.05 compared to respective untreated control.

FIG. 11 is a bar graph showing the densitometeric analysis of a WesternBlot analysis of HbF protein expression in AA and SS primary RPE. Thegraph is γ-Globin/β-Actin (Relative Densitometry) in cells treated withMMF or HU. (UT, untreated).

FIG. 12 is a bar graph of OCTN1 mRNA Expression (Fold Change) in KU812cells treated with 1000 μM MMF for 16 hours. Control cells areidentified as CON. Data are represented as mean+standard error of themean; *p<0.05.

FIG. 13 is bar graph of OCTN1 mRNA Expression (Fold Change) in ARPE-19cells treated with 1000 μM MMF, 100 μm HU, or 1000 μM MMF and 100 μM HU.

FIG. 14 is a bar graph of mOCTN1 mRNA expression (Fold Change) in AA orSS cells treated with 1000 μM MMF.

FIG. 15 is a bar graph of OCTN1 mRNA expression (Fold Change) in primarymouse RPE (pRPE), Muller (pMC) and ganglion (pGC) cells.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The term “expression control sequence” refers to a nucleic acid sequencethat controls and regulates the transcription and/or translation ofanother nucleic acid sequence. Control sequences that are suitable forprokaryotes, for example, include a promoter, optionally an operatorsequence, a ribosome binding site, and the like. Eukaryotic cells areknown to utilize promoters, polyadenylation signals, and enhancers.

The term “gene” refers to a DNA sequence that encodes through itstemplate or messenger RNA a sequence of amino acids characteristic of aspecific peptide, polypeptide, or protein. The term “gene” also refersto a DNA sequence that encodes an RNA product. The term gene as usedherein with reference to genomic DNA includes intervening, non-codingregions as well as regulatory regions and can include 5′ and 3′ ends.

As generally used herein “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues, organs, and/or bodily fluids of human beings andanimals without excessive toxicity, irritation, allergic response, orother problems or complications commensurate with a reasonablebenefit/risk ratio.

The terms “subject,” “individual,” and “patient” refer to any individualwho is the target of treatment using the disclosed compositions. Thesubject can be a vertebrate, for example, a mammal. Thus, the subjectcan be a human. The subjects can be symptomatic or asymptomatic. Theterm does not denote a particular age or sex. Thus, adult and newbornsubjects, whether male or female, are intended to be covered. A subjectcan include a control subject or a test subject. The test subject can bea subject afflicted with a genetic mutation in the beta-globin gene oran expression control sequence thereof, or a subject with a sickle celldisorder, a globinopathy, or a beta-thalassemia.

As used herein, the term “treating” includes alleviating the symptomsassociated with a specific disorder or condition and/or preventing oreliminating said symptoms.

The term “alkyl” refers to the radical of saturated aliphatic groups,including straight-chain alkyl groups, branched-chain alkyl groups,cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, andcycloalkyl-substituted alkyl groups.

In preferred embodiments, a straight chain or branched chain alkyl has30 or fewer carbon atoms in its backbone (e.g., C1-C30 for straightchains, C3-C30 for branched chains), preferably 20 or fewer, morepreferably 15 or fewer, most preferably 10 or fewer. Likewise, preferredcycloalkyls have from 3-10 carbon atoms in their ring structure, andmore preferably have 5, 6, or 7 carbons in the ring structure. The term“alkyl” (or “lower alkyl”) as used throughout the specification,examples, and claims is intended to include both “unsubstituted alkyls”and “substituted alkyls”, the latter of which refers to alkyl moietieshaving one or more substituents replacing a hydrogen on one or morecarbons of the hydrocarbon backbone. Such substituents include, but arenot limited to, halogen, hydroxyl, carbonyl (such as a carboxyl,alkoxycarbonyl, formyl, or an acyl), thiocarbonyl (such as a thioester,a thioacetate, or a thioformate), alkoxyl, phosphoryl, phosphate,phosphonate, phosphinate, amino, amido, amidine, imine, cyano, nitro,azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl,sulfonamide, sulfonyl, heterocyclyl, aralkyl, or an aromatic orheteroaromatic moiety.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto ten carbons, more preferably from one to six carbon atoms in itsbackbone structure. Likewise, “lower alkenyl” and “lower alkynyl” havesimilar chain lengths. Throughout the application, preferred alkylgroups are lower alkyls. In preferred embodiments, a substituentdesignated herein as alkyl is a lower alkyl.

It will be understood by those skilled in the art that the moietiessubstituted on the hydrocarbon chain can themselves be substituted, ifappropriate. For instance, the substituents of a substituted alkyl mayinclude halogen, ulfonam, nitro, thiols, amino, azido, imino, amido,phosphoryl (including phosphonate and phosphinate), sulfonyl (includingsulfate, ulfonamide, sulfamoyl and sulfonate), and silyl groups, as wellas ethers, alkylthios, carbonyls (including ketones, aldehydes,carboxylates, and esters), —CF3, —CN and the like. Cycloalkyls can besubstituted in the same manner.

“Aryl”, as used herein, refers to C5-C10-membered aromatic,heterocyclic, fused aromatic, fused heterocyclic, biaromatic, orbihetereocyclic ring systems. Broadly defined, “aryl”, as used herein,includes 5-, 6-, 7-, 8-, 9-, and 10-membered single-ring aromatic groupsthat may include from zero to four heteroatoms, for example, benzene,pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole,pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like.Those aryl groups having heteroatoms in the ring structure may also bereferred to as “aryl heterocycles” or “heteroaromatics”. The aromaticring can be substituted at one or more ring positions with one or moresubstituents including, but not limited to, halogen, azide, alkyl,aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino (orquaternized amino), nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,ulfonamide, ketone, aldehyde, ester, heterocyclyl, aromatic orheteroaromatic moieties, —CF3, —CN; and combinations thereof.

The term “aryl” also includes polycyclic ring systems having two or morecyclic rings in which two or more carbons are common to two adjoiningrings (i.e., “fused rings”) wherein at least one of the rings isaromatic, e.g., the other cyclic ring or rings can be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls and/or heterocycles. Examples ofheterocyclic rings include, but are not limited to, benzimidazolyl,benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl,benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl,benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aHcarbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl,decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl,dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl,imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl,indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl,isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl,isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl,naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl,oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl,piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl,pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl,pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole,pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl,pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl,quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl,tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl,1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl,1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl,thienooxazolyl, thienoimidazolyl, thiophenyl and xanthenyl. One or moreof the rings can be substituted as defined above for “aryl”.

II. Methods of Treating Sickle Cell Disease, Beta-Thalassemias, andRelated Disorders

A. Treatment of SCD with Fumaric Acid Esters

Methods of increasing expression of HbF in cells by contacting thecells, for example erythroid and RPE cells, with an effective amount ofa fumaric acid ester, or pharmacologically active salt, derivative,analogue or prodrug thereof are disclosed. The methods can be used tocompensate for a mutation in the human beta-globin gene in cells thathave one or more mutations in the beta-globin gene or an expressioncontrol sequence thereof, for example mutations that result in theexpression of the Hb S form of hemaglobin. Compensating for the mutationincludes but is not limited to increasing the amount of HbF and reducingthe amount of Hb S in the subject compared to untreated subjects. Themethods can be used for treating sickle cell disease, for example sicklecell anemia, and other hemoglobinopathies or thalassemias as well ascomplications related to SCD, for example retinopathy.

B. Treatment of Fumaric Acid Esters in Combination or Alternation withHU.

Methods for treating SCD or complications thereof include administeringfumaric acid esters in combination or alternation with HU in amountseffective to induce or increase expression of HbF and increaseexpression of OCTN1 in erythroid and retinal cells. It has beendiscovered that MMF induce the expression of SLC22A4 (aka OCTN1), atransporter shown recently to transport HU (Walker A L et al. Exp.Hematol. 2011; 39(4):446-56). The expression of OCTN1 and its inductionby MMF is evident in retinal and erythroid cells. MMF can increase theentry of HU into erythroid progenitor cells and facilitate the action ofHU on fetal hemoglobin production. In some embodiments, MMF can reducethe dosing of HU in SCD patients without compromising its therapeuticefficacy, and reduce the toxic side effects associated with HU therapy.

Subjects with SCD that are unresponsive to HU treatment can be treatedby administering a fumaric acid ester in combination or alternation withHU. While MMF adjuvant therapy along with HU would certainly benefit SCDpatients who respond to HU, it also has potential to work on those whodo not respond to HU. In one embodiment, the “non-responders” expresslower levels of OCTN1 than “responders.” The decreased expression of thetransporter would result in decreased entry of HU into its target cells(erythroid progenitors) and thus decrease its pharmacological effect.Since MMF induces OCTN1, adjuvant therapy is likely to enhance the entryof HU in ‘non-responders” and thereby make the patients to becomeresponsive to HU therapy. This is in addition to the effect of MMFitself in increasing fetal hemoglobin production.

Fumaric acid esters have been used for greater than 50 years in thetreatment of psoriasis, and more recently multiple sclerosis. It isbelieved that the beneficial effects of fumaric acid esters in thetreatment of these pathologic conditions are due to their potentanti-inflammatory and anti-oxidant effects. It has been discovered thatin addition to the known, robust anti-inflammatory and anti-oxidantproperties, these compounds also induce production of fetal hemoglobin(HbF) in cells. Reactivation of HbF, which is typically absent orexpressed only at low levels in humans over six months of age, isconsidered a viable approach for treating children and adults withsickle cell disease, and other hemoglobinopathies and thalassemias. Themethods disclosed herein typically include administering a fumaric acidester, or pharmacologically active salt, derivative, analogue or prodrugthereof to a subject in need thereof to increase expression of HbF inthe subject, to increase expression of OCTN1, or both.

C. Diseases to be Treated

The disclosed compositions can be used to treat subjects with one ormore mutations in the beta-globin gene (HBB gene). Mutations in the betaglobin gene can cause sickle cell disease, beta thalassemia, or relateddiseases or conditions thereof. As discussed in more detail below,mutations in the beta-globin gene can be identified before or aftermanifestations of a disease's clinical symptoms. The compositions can beadministered to a subject with one or more mutations in the beta-globingene before or after the onset of clinical symptoms. Therefore, in someembodiments, the compositions are administered to a subject that hasbeen diagnosed with one or more mutations in the beta-globin gene, butdoes not yet exhibit clinical symptoms. In some embodiments, thecompositions are administered to a subject that is exhibiting one ormore symptoms of a disease, condition, or syndrome associated with, orcaused by one or more mutations in the beta-globin gene.

1. Sickle Cell Disease

Sickle cell disease (SCD) typically arises from a mutation substitutingthymine for adenine in the sixth codon of the beta-chain gene ofhemoglobin (i.e., GAG to GTG of the HBB gene). This mutation causesglutamate to valine substitution in position 6 of the Hb beta chain. Theresulting Hb, referred to as HbS, has the physical properties of formingpolymers under deoxy conditions. SCD is typically an autosomal recessivedisorder. Therefore, in some embodiments, the disclosed compositions andmethods are used to treated a subject homozygous for an autosomalrecessive mutation in beta-chain gene of hemoglobin (i.e., homozygousfor sickle cell hemoglobin (HbS)). Also referred to as HbSS disease orsickle cell anemia (the most common form), subjects homozygote for the Sglobin typically exhibit a severe or moderately severe phenotype andhave the shortest survival of the hemaglobinopathies.

Sickle cell trait or the carrier state is the heterozygous formcharacterized by the presence of around 40% HbS, absence of anemia,inability to concentrate urine (isosthenuria), and hematuria. Underconditions leading to hypoxia, it may become a pathologic risk factor.Accordingly, in some embodiments, the disclosed compositions and methodsare used to treat a subject heterozygous for an autosomal recessivemutation in the beta-chain gene of hemoglobin (i.e., heterozygous forHbS).

2. Beta-Thalassemia

Beta-thalassemias (β-thalassemias) are a group of inherited blooddisorders caused by a variety of mutational mechanisms that result in areduction or absence of synthesis of β-globin and leading toaccumulation of aggregates of unpaired, insoluble α-chains that causeineffective erythropoiesis, accelerated red cell destruction, and severeanemia. Subjects with beta-thalassemia exhibit variable phenotypesranging from severe anemia to clinically asymptomatic individuals. Thegenetic mutations present in β thalassemias are diverse, and can becaused by a number of different mutations. The mutations can involve asingle base substitution or deletions or inserts within, near orupstream of the β globin gene. For example, mutations occur in thepromoter regions preceding the beta-globin genes or cause production ofabnormal splice variants.

Examples of thalassemias include thalassemia minor, thalassemiaintermedia, and thalassemia major.

Thalassemia minor refers to thalassemia where only one of beta-globinalleles bears a mutation. Individuals typically suffer from microcyticanemia. Detection usually involves lower than normal MCV value (<80 fL)plus an increase in fraction of Hemoglobin A2 (>3.5%) and a decrease infraction of Hemoglobin A (<97.5%). Genotypes can be β+/β or β-0/β.

Thalassemia intermedia refers to a thalassemia intermediate between themajor and minor forms. Affected individuals can often manage a normallife but may need occasional transfusions, e.g., at times of illness orpregnancy, depending on the severity of their anemia. Genotypes can beβ+/β+ or β-0/β.

Thalassemia major refers to a thalassemia where both beta-globin alleleshave thalassemia mutations. This is a severe microcytic, hypochromicanemia. If left untreated, it causes anemia, splenomegaly, and severebone deformities and typically leads to death before age 20. Treatmentconsists of periodic blood transfusion; splenectomy if splenomegaly ispresent, and treatment of transfusion-caused iron overload. Cure ispossible by bone marrow transplantation. Cooley's anemia is named afterThomas Benton Cooley. Genotypes include β+/β-0 or β-0/β-0 or β+/β+.

3. Sickle Cell Related Disorders

Although carriers of sickle cell trait do not suffer from SCD,individuals with one copy of HbS and one copy of a gene that codes foranother abnormal variant of hemoglobin, such as HbC or Hbbeta-thalassemia, have a less severe form of the disease. For example,another specific defect in beta-globin causes another structuralvariant, hemoglobin C (HbC). Hemoglobin C (abbreviated as Hb C or HbC)is an abnormal hemoglobin in which substitution of a glutamic acidresidue with a lysine residue at the 6^(th) position of the β-globinchain has occurred. A subject that is a double heterozygote for HbS andHbC (HbSC disease) is typically characterized by symptoms of moderateclinical severity.

Another common structural variant of beta-globin is hemoglobin E orhemoglobin E (HbE). HbE is an abnormal hemoglobin in which substitutionof a glutamic acid residue with a lysine residue at the 26^(th) positionof the β-globin chain has occurred. A subject that is a doubleheterozygote for HbS and HbE has HbS/HbE syndrome, which usually causesa phenotype similar to HbS/b+ thalassemia, discussed below.

Some mutations in the beta-globin gene can cause other structuralvariations of hemoglobin or can cause a deficiency in the amount ofβ-globin being produced. These types of mutations are referred to asbeta-thalassemia mutations.

The absence of beta-globin is referred to as beta-zero (β-0)thalassemia. A subject that is a double heterozygote for HbS and β-0thalassemia (i.e., HbS/β-0 thalassemia) can suffer symptoms clinicallyindistinguishable from sickle cell anemia.

A reduced amount of beta-globin is referred to as β-plus (β+)thalassemia. A subject that is a double heterozygote for HbS and β+thalassemia (i.e., HbS/β+ thalassemia) can have mild-to-moderateseverity of clinical symptoms with variability among differentethnicities.

Rare combinations of HbS with other abnormal hemoglobins include HbD LosAngeles, G-Philadelphia, HbO Arab, and others.

Therefore, in some embodiments, the disclosed compositions and methodsare used to treating a subject with an HbS/β-0 genotype, an HbS/β+genotype, an HBSC genotype, an HbS/HbE genotype, an HbD Los Angelesgenotype, a G-Philadelphia genotype, or an abHbO Arab genotype.

As discussed above, retinopathy due to SCD can also be treated byadministering an effective amount of a fumaric acid ester, for exampleMMF, optionally in combination or alternation with HU in amountseffective to induce expression of HbF in retinal cells, for example inRPE cells. Sickle retinopathy occurs when the retinal blood vessels getoccluded by sickle red blood cells and the retina becomes ischemic,angiogenic factors are made in retina. In sickle cell disease, thisoccurs mostly in the peripheral retina, which does not obscure vision atfirst. Eventually, the entire peripheral retina of the sickle cellpatient becomes occluded and many neovascular formations occur.Administration of one or more fumaric acid esters optionally incombination with HU can reduce or inhibit the formation of occlusions inthe peripheral retina of a sickle cell patient.

4. Non-Erythroid Cell Related Disorders

Although red blood cells are the primary producers of hemoglobin,reports indicate that other, non-hematopoietic cells, including, but notlimited to, macrophage, retinal pigment cells, and alveolar epithelialcells such as alveolar type II (ATII) cells and Clara cells which arethe primary producers of pulmonary surfactant, also synthesizehemoglobin (Newton, et al., J. Biol. Chem., 281(9)5668-5676 (2006),Tezel, et al., Invest. Ophthalmol. Vis. Sci., 50(4):1911-9 (2009), Liu,et al., Proc. Natl. Acad. Sci. USA, 96(12)6643-6647 (1999)). Thesefindings are consistent with the conclusion that the expression ofhemoglobin by non-erythroid cells at interfaces where oxygen-carbondioxide diffusion occurs may be an adaptive mechanism to facilitateoxygen transport (Tezel, et al., Invest. Ophthalmol. Vis. Sci.,50(4):1911-9 (2009).

Therefore, in some embodiments, the compositions disclosed herein areused to increase HbF expression in non-erythroid cells including, butnot limited to, macrophage, retinal pigment cells, and alveolarepithelial cells such as alveolar type II (ATII) cells and Clara cells.In some embodiments, the compositions disclosed herein are used toincrease HbF expression in non-erythroid cells at interfaces whereoxygen-carbon dioxide diffusion occurs, including, but not limited tothe eyes and lungs. In some embodiments, the compositions are used toinduce, increase, or enhance hemoglobin synthesis retinal pigment cellsin an effective amount to prevent, reduce, or alleviate one or moresymptoms of age-related macular degeneration or diabetic retinopathy.

D. Symptoms of Sickle Cell Disease, Beta-Thalassemias, and RelatedDisorders

In some embodiments, the compositions disclosed herein are administeredto a subject in an effective amount to treatment one or more symptoms ofsickle cell disease, a beta-thalassemia, or a related disorder.

Beta-thalassemia can include symptoms such as anemia, fatigue andweakness, pale skin or jaundice (yellowing of the skin), protrudingabdomen with enlarged spleen and liver, dark urine, abnormal facialbones and poor growth, and poor appetite.

In subjects with sickle cell disease, or a related disorder,physiological changes in RBCs can result in a disease with the followingsigns: (1) hemolytic anemia; (2) vaso-occlusive crisis; and (3) multipleorgan damage from microinfarcts, including heart, skeleton, spleen, andcentral nervous system.

Chronic Hemolytic Anemia

SCD is a form of hemolytic anemia, with red cell survival of around10-20 days. Approximately one third of the hemolysis occursintravascularly, releasing free hemoglobin (plasma free hemoglobin[PFH]) and arginase into plasma. PFH has been associated withendothelial injury including scavenging nitric oxide (NO),proinflammatory stress, and coagulopathy, resulting in vasomotorinstability and proliferative vasculopathy. A hallmark of thisproliferative vasculopathy is the development of pulmonary hypertensionin adulthood.

Vaso-Occlusive Crisis

Vaso-occlusive crisis occurs when the circulation of blood vessels isobstructed by sickled red blood cells, causing ischemic injuries. Themost common complaint is of pain, and recurrent episodes may causeirreversible organ damage. One of the most severe forms is the acutechest syndrome which occurs as a result of infarction of the lungparenchyma. Vaso-occlusive crisis can be accompanied by a pain crisiswhich can occur suddenly and last several hours to several days.

The pain can affect any body part. It often involves the abdomen, bones,joints, and soft tissue, and it may present as dactylitis (bilateralpainful and swollen hands and/or feet in children), acute joint necrosisor avascular necrosis, or acute abdomen. With repeated episodes in thespleen, infarctions and autosplenectomy predisposing to life-threateninginfection are usual. The liver also may infarct and progress to failurewith time. Papillary necrosis is a common renal manifestation ofvaso-occlusion, leading to isosthenuria (i.e, inability to concentrateurine).

Severe deep pain is present in the extremities, involving long bones.Abdominal pain can be severe, resembling acute abdomen; it may resultfrom referred pain from other sites or intra-abdominal solid organ orsoft tissue infarction. Reactive ileus leads to intestinal distentionand pain.

Bone pain and abdominal pain may be present. The face also may beinvolved. Pain may be accompanied by fever, malaise, and leukocytosis.

Skeletal Manifestations

Skeletal manifestations include, but are not limited to, infarction ofbone and bone marrow, compensatory bone marrow hyperplasia, secondaryosteomyelitis, secondary growth defects, intravascular thrombosis,osteonecrosis (avascular necrosis/aseptic necrosis), degenerative boneand joint destruction, osteolysis (in acute infarction), Articulardisintegration, myelosclerosis, periosteal reaction (unusual in theadult), H vertebrae (steplike endplate depression also known as theReynold sign or codfish vertebrae), Dystrophic medullary calcification,bone-within-bone appearance, decreased density of the skull, decreasedthickness of outer table of skull due to widening of diploe, hair on-endstriations of the calvaria, osteoporosis sometimes leading to biconcavevertebrae, coarsening of trabeculae in long and flat bones, andpathologic fractures, bone shortening (premature epiphyseal fusion),epiphyseal deformity with cupped metaphysis, peg-in-hole defect ofdistal femur, and decreased height of vertebrae (short stature andkyphoscoliosis).

Renal Manifestations

Renal manifestations include, but are not limited to, various functionalabnormalities such as hematuria, proximal tubule dysfunction, impairedpotassium excretion, and hyperkalemia; and gross anatomic alterations,for example, hypertrophied kidneys, with a characteristic smooth,capsular surface.

Splenic Manifestations

Splenic manifestations include, but are not limited to, enlargement,including rapid and/or painful enlargement known as splenicsequestration crisis, infarction, low pH and low oxygen tension in thesinusoids and splenic cords, functional impairment, autosplenectomy(fibrosis and shrinking of the spleen in advanced cases), immunedeficiency and increased risk of sepsis.

Other Common Symptoms

Lower serum immunoglobulin M (IgM) levels, impaired opsonization, andsluggish alternative complement pathway activation, increasesusceptibility to infection pneumonia, bronchitis, cholecystitis,pyelonephritis, cystitis, osteomyelitis, meningitis, and sepsis andother challenges from infectious agents including, but not limited to,Mycoplasma pneumoniae, Salmonella typhimurium, Staphylococcus aureus,and Escherichia coli; growth delays or maturation delays during pubertyin adolescents, hand-foot syndrome, acute chest syndrome, stroke,hemiparesis, hemosiderin deposition in the myocardium, dilation of bothventricles and the left atrium, cholelithiasis, paraorbital facialinfarction, retinal vascular changes, proliferative retinitis, loss ofvision, leg ulcers, priapism, avascular necrosis, and pulmonaryhypertension.

III. Compositions for Use in Treating Sickle Cell Disease,Beta-Thalassemia, or Related Disorders

A. Active Agents

1. Fumaric Acid Esters

The methods disclosed herein typically include administering a subjectin need thereof one or more fumaric acid esters or pharmacologicallyactive salts, derivatives, analogues or prodrugs thereof. In preferredembodiments, the one or more fumaric acid esters, pharmacologicallyactive salts, derivatives, analogues, or prodrugs thereof are part ofpharmaceutical compositions which can include a pharmaceuticallyacceptable carrier. Fumaric acid esters (FAE) are agents derived fromthe unsaturated dicarbonic acid, fumaric acid. Fumaric acid is a whitecrystalline powder with a characteristic acidic taste that is commonlyused as a food additive and flavoring agent in cakes and sweets. Fumaricacid is poorly absorbed and believed to pass through the body withoutcausing any effects. However, esters of fumaric acid (FAEs) are potentchemicals and recognized for their ability to treat clinical symptoms ofpsoriasis and multiple sclerosis.

In one embodiment, the fumaric acid ester has the following formula:

wherein R and R′ are independently selected from the group consisting ofhydrogen or substituted or unsubstituted alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, alkenyl, heteroalkenyl, cycloalkenyl,heterocycloalkenyl, aryl, and heteroaryl, with the provision that R andR′ are not both hydrogen.

In some embodiments, one or both of R and R′ are lower alkyl (e.g.,C₁-C₄), such as substituted or unsubstituted methyl or ethyl. ExemplaryFAEs include, but are not limited to, monoethyl fumarate (MEF),monomethyl fumarate (MMF), diethyl fumarate (DEF), dimethyl fumarate(DMF), as well as pharmacologically active salts, derivatives,analogues, or prodrugs thereof.

Relationships between the physicochemical properties of the fumaric acidesters, including their presystemic metabolism and intestinalabsorption, are known in the art. See, for example, Werdenberg, et al.,Biopharm. Drug Dispos., 24(6):259-73 (2003), which reports that theintestinal permeability of the monoesters methyl hydrogen fumarate,ethyl hydrogen fumarate, n-propylhydrogen fumarate and n-pentyl hydrogenfumarate increase with an increase in their lipophilicity, however,their presystemic metabolism rates likewise increase with increasingester chain length. Therefore, it is believed that for fumarates, anincrease in intestinal permeability of the more lipophilic derivativesis counterbalanced by an increase in first-pass extraction.

Additional studies on characterizing the intestinal absorption offumaric acid esters indicate that uncharged diester dimethylfumaratedisplays a high presystemic metabolic liability and high permeability inan in vitro small intestinal cell model (Werdenberg, et al., Biopharm.Drug Dispos., 24(6):259-73 (2003)). Results also show completemetabolism of DFM in the intestinal tissue.

DMF is rapidly hydrolysed by esterases to the metabolite MMF.Accordingly, in a preferred embodiment the fumaric acid ester is DMF,MMF, or a combination thereof. In some embodiments, the compositionsalso includes Monoethyl fumarate.

Formulations including dimethyl fumarate and ethyl hydrogen fumaratehave been used in the treatment of psoriasis for many years. One familyof such formulations are marketed under the tradename FUMADERM. FUMADERMis in the form of tablets intended for oral use and it is available intwo different dosage strengths (FUMADERM Initial and FUMADERM):

TABLE 1 Components and Quantitative Composition of FUMADERM (U.S. patentapplication 2008/0004344) Fumaderm ® Initial Fumaderm ® Dimethylfumarate30 mg 120 mg  Ethylhydrogenfumarate, 67 mg 87 mg  calcium saltEthylhydrogenfumarate,  5 mg 5 mg Magnesium salt Etylhydrogenfumarate, 3 mg 3 mg Zinc salt

For the treatment of psoriasis, the two strengths are typically appliedin an individually based dose regimen starting with FUMADERM Initial inan escalating dose, and then after e.g., three weeks of treatmentswitching to FUMADERM. Both FUMADERM Initial and FUMADERM are entericcoated tablets. In some embodiments, the composition used in the methodsdisclosed herein includes FUMADERM Initial, FUMADERM, or a combinationthereof.

Another marketed composition is FUMARAAT 120 which contains 120 mg ofdimethylfumarate and 95 mg of calcium monoethylfumarate (TioFarma,Oud-Beijerland, Netherlands). In the publication (Litjens et al. Br. J.Clin. Pharmacol. 2004, vol. 58:4, pp. 429-432), the pharmacokineticprofile of FUMARAAT 120 was reported in healthy subjects. The resultsshow that a single oral dose of FUMARAAT 120 is followed by a rise inserum monomethylfumarate concentration and only negligibleconcentrations of dimethylfumarate and fumaric acid is observed. Theresults indicate that dimethylfumarate is rapidly hydrolyzed tomonomethylfumarate in an alkaline environment, but according to theauthors not in an acid environment. As the composition is entericcoated, it is believed that the uptake of fumarate takes place mainly inthe small intestine. It is believed that dimethylfumarate is eitherhydrolysed to the monoester before uptake due to an alkaline environmentor it is rapidly converted to monoester by esterases in the circulation.In some embodiments, the composition used in the methods disclosedherein includes FUMARAAT 120.

The study also shows that time to peak concentration (T_(max)) and peakconcentration (C_(max)) are subject to food effect, i.e., T_(max) isprolonged (mean for fasted conditions is 182 min, whereas for fedconditions mean is 361 min) [lag time is 90 min for fasted and 300 minfor fed] and C_(max) is decreased (fasted: 0.84 mg/l, fed: 0.48 mg/l) byconcomitant food-intake.

Another study, in healthy subjects with two tablets of FUMADERM,revealed C. values (determined as monoethyl- or monomethylfumarate) in arange from 1.0 to 2.4 μg/ml and a T_(max) in a range of from 4.8 to 6.0hours (Reddingius W. G. Bioanalysis and Pharmacokinetics of Fumarates inHumans. Dissertation ETH Zurich No. 12199 (1997)).

U.S. Published Application 2012/0165404, which is specificallyincorporated by reference herein in its entirety, describes compositionsreferred to as BG00012, an orally available formulation of dimethylfumarate (DMF) which is in clinical development for treatment ofrelapsing-remitting multiple sclerosis (RRMS). U.S. PublishedApplication 2012/0165404, describes that some embodiments in whichdimethyl fumarate is administered to a patient the DMF is formulated incapsules containing enteric coated microtablets referred to “BG-12” or“BG00012.” The coating of the tablets is composed of different layers.The first layer is a methacrylic acid-methyl methacrylatecopolymer/isopropyl alcohol solution which isolates the tablet coresfrom potential hydrolysis from the next applied water suspensions.Enteric coating of the tablet is then conferred by an aqueousmethacrylic acid-ethyl acrylate copolymer suspension. In someembodiments, the composition used in the methods disclosed hereinincludes BG00012. The complete components and quantitative compositionof the capsules are given in Table 2.

TABLE 2 Components and Quantitative Composition of BG00012, (U.S. patentapplication No. 2012/0165404) Ingredients Amount/capsule Function CoreMicrotablets Active ingredients: Dimethyl Fumarate* 120.00 mg activeingredient Excipients: Croscarmellose sodium  15.00 mg disintegrantMicrocrystalline Cellulose 131.60 mg filler Magnesium stearate  5.00 mglubricant Talcum  19.80 mg glidant Silica colloidal anhydrous  2.60 mgglidant Mass core microtablets 294.00 mg Coating MicrotabletsExcipients: Triethyl Citrate**  7.60 mg plasticizer MethacrylicAcid-Methyl  5.50 mg film coating agent Methacrylate Copolymer (1:1) asMethacrylic Acid-Methyl  (44.00 mg) Methacrylate Copolymer (1:1)solution 12.5%** Simeticone (corresponding to  0.17 mg anti-foam agentSimeticone Ph Eur) as Simeticone Emulsion USP**  (0.53 mg) Talcummicronised**  13.74 mg lubricant Methacrylic acid - Ethyl Acrylate 33.00 mg film coating agent Copolymer (1:1) as Methacrylic acid - Ethyl(110.00 mg) Acrylate Copolymer (1:1) dispersion 30% ** Mass entericcoated microtablets 354.01 mg Mass of gelatin capsule  96.00 mg Mass offilled capsule 450.01 mg

In some embodiments, the fumaric acid ester is a prodrug of a fumaricacid ester. Preliminary results from a Phase 1 clinical trial in healthyadults designed to assess the pharmacokinetics (PK), safety andtolerability of single doses of four different oral formulations of afumaric acid ester compound that is a prodrug of monomethyl fumarate(MMF) referred to as XP23829 were favorable (XenoPort Press Release,“XenoPort Reports Favorable Metabolism and Pharmacokinetics of XP23829,a Novel Fumaric Acid Ester, in Phase 1 Trial” (2012)). XP23829 is beingdeveloped for the potential treatment of relapsing-remitting multiplesclerosis (RRMS) and/or psoriasis. The trial showed that administrationof XP23829 resulted in the expected levels of MMF in the blood. The fourformulations produced different PK profiles of MMF, including oneformulation that could potentially be dosed two or three times a day andat least one formulation that may be suitable for once-a-day dosing.XP23829 was generally well-tolerated in the trial. U.S. PatentApplication Nos. 2012/0157523, 2012/0095003, and 2010/00048651, each ofwhich is incorporated by reference in its entirety, discuss prodrugs ofmethyl hydrogen fumarate, pharmaceutical compositions thereof andmethods of use. In some embodiments, the fumaric acid ester is a fumaricacid ester prodrug such as XP23829, or pharmacologically active salt, ananalogue or derivative thereof.

Other suitable fumaric acid ester compounds, pharmaceuticalcompositions, and formulations suitable for use in the disclosed methodsare known in the art. See for example, U.S. Pat. Nos. 6,509,376,6,436,992, 6,277,882, 6,355,676, 6,509,376, 4,959,389, and U.S. PatentApplication No. 2008/0004344, each of which is incorporated by referencein its entirety. U.S. Pat. Nos. 6,509,376 and 6,436,992 discussformulations containing DMF and/or MMF. U.S. Pat. Nos. 6,277,882 and6,355,676 describe the use of alkyl hydrogen fumarates and certainfumaric acid mono alkyl ester salts, respectively, for preparing microtablets for treating psoriasis, psoriatic arthritis, neurodermatitis andenteritis regionalis. U.S. Pat. No. 6,509,376 describes the use ofcertain dialkyl fumarates pharmaceutical preparations for use intransplantation medicine or the therapy of autoimmune diseases in theform of micro tablets or pellets, U.S. Pat. No. 4,959,389, whichdescribes compositions containing different salts of fumaric acidmonoalkyl ester alone or in combination with dialkyl fumarate, and U.S.Patent Application No. 2008/0004344, which describes salts of fumaricacid monoalkylesters and their pharmaceutical use. The Case report“Treatment of disseminated granuloma annulare with fumaric acid esters”from BMC Dermatology, vol. 2, no. 5, 2002, relates to treatment withfumaric acid esters.

2. Co-Administration

The compositions disclosed herein can optionally include, or beco-administered with one or more additional active agents.Co-administration can include the simultaneous and/or sequentialadministration of the one or more additional active agents and one ormore fumaric acid ester, or pharmacologically active salt, derivative,analogue, or prodrug thereof. The one or more additional active agentsand the fumaric acid ester, or pharmacologically active salt,derivative, analogue, or prodrug thereof can be included in the same ordifferent pharmaceutical formulation. The one or more additional activeagents and the fumaric acid ester, or pharmacologically active salt,derivative, analogue, or prodrug thereof can achieve the same ordifferent clinical benefit. An appropriate time course for sequentialadministration may be chosen by the physician, according to such factorsas the nature of a patient's illness, and the patient's condition. Incertain embodiments, sequential administration includes theco-administration of one or more additional active agents and thenanoparticle gene carriers within a period of one week, 72 hours, 48hours, 24 hours, or 12 hours.

The additional active agent can be chosen by the user based on thecondition or disease to be treated. Example of additional active agentsinclude, but are not limited to, vitamin supplements, nutritionalsupplements, anti-anxiety medication, anti-depression medication,anti-coagulants, clotting factors, anti-inflammatories, steroids such ascorticosteroids, analgesic, etc.

In some embodiments, the compositions disclosed herein areco-administered in combination with one or more additional active agentsfor treatment of sickle cell disease, beta-thalassemia, or a relateddisorder. Such additional active agents may include, but are not limitedto, folic acid, penicillin or another antibiotics, preferably aquinolone or macrolide, antivirals, anti-malarial prophylactics, andanalgesics to control pain crises.

In some embodiments, the compositions are co-administered with one ormore additional agents that increase expression of HbF, for example,hydroxyurea.

In some embodiments, the compositions are co-administered with one ormore additional treatment protocols, for example, transfusion therapy,stem cell therapy, gene therapy, bone marrow transplants, dialysis orkidney transplant for kidney disease, gallbladder removal in people withgallstone disease, hip replacement for avascular necrosis of the hip,surgery for eye problems, and wound care for leg ulcers.

B. Effective Amounts

In some embodiments, the compositions are administered in an amounteffective to induce a pharmacological, physiological, or moleculareffect compared to a control that is not administered the composition.In some embodiments, a fumaric acid ester, or pharmacologically activesalt, derivative, analogue, or prodrug thereof is administered to asubject in need thereof to increase expression of HbF in the subject.For example, HbF expression can be increased in an amount effective tocompensate for, or reduce the effects of a mutation in the HBB gene. Insome embodiments, the fumaric acid ester, or pharmacologically activesalt, derivative, analogue, or prodrug thereof is administered in aneffective amount to reduce the sickling of red blood cells in a patientrelative to a control.

In some embodiments, the fumaric acid ester, or pharmacologically activesalt, derivative, analogue, or prodrug thereof is provided in aneffective amount to prevent, reduce or alleviate one or more symptoms ofa disease or disorder to be treated. For example, the compositionsdisclosed herein can be administered to a subject in need thereof in aneffective amount to reduce or alleviate one or more symptoms of sicklecell disease, a beta-thalassemia, or a sickle cell related disorder,including, but not limited to, the symptoms discussed above.

Suitable controls are known in the art and can be determined based onthe disease to be treated. Suitable controls include, but are notlimited to a subject, or subjects without sickle cell disease, abeta-thalassemia, or a sickle cell related disorder; or a condition orstatus of a subject with the disease or disorder prior to initiation ofthe treatment. For example, in some embodiments, treatment of a subjectwith a fumaric acid ester, or pharmacologically active salt, derivative,analogue, or prodrug thereof improves one or more pharmacological,physiological, or molecular effects; reduces or alleviates one or moresymptoms of the disease or disorder to be treated; or a combinationthereof compared to a subject or subjects without the disease ordisorder to be treated. In some embodiments, treatment of a subject witha fumaric acid ester, or pharmacologically active salt, derivative,analogue, or prodrug thereof improves one or more pharmacological,physiological, or molecular effects; reduces or alleviates one or moresymptoms of the disease or disorder to be treated; or a combinationthereof in the subject compared to the same pharmacological,physiological, or molecular effects; or symptoms of the disease ordisorder in the subject prior to administration of the fumaric acidester, or pharmacologically active salt, derivative, analogue, orprodrug thereof to the subject.

In some embodiments, the fumaric acid ester, or pharmacologically activesalt, derivative, analogue, or prodrug thereof is administered to asubject in need thereof in an effective amount to improve one or morepharmacological, physiological, or molecular effects, or to reduce oralleviate one or more symptoms of the disease or disorder with higherefficacy, lower toxicity, or a combination thereof compared to a subjecttreated with an different therapeutic agent such as hydroxyurea (HU).

C. Dosages and Dosage Regimes

For all of the disclosed compounds, as further studies are conducted,information will emerge regarding appropriate dosage levels fortreatment of various conditions in various patients, and the ordinaryskilled worker, considering the therapeutic context, age, and generalhealth of the recipient, will be able to ascertain proper dosing. Theselected dosage depends upon the desired therapeutic effect, on theroute of administration, and on the duration of the treatment desired.Generally dosage levels of 0.001 to 100 mg/kg of body weight daily areadministered to mammals. Generally, for intravenous injection orinfusion, dosage may be lower.

As discussed above some fumaric acid esters have been administered totreat patients for psoriasis and multiple sclerosis.

For example, an exploratory, prospective, open-label study of fumaricacid esters (FAE, FUMADERM) was conducted in patients withrelapsing-remitting multiple sclerosis. The study consisted of thefollowing four phases: 6-week baseline, 18-week treatment (target doseof 720 mg/day), 4-week washout, and a second 48-week treatment phase(target dose of 360 mg/day) (Schimrigk, et al., Eur. J. Neurol.,13(6):604-10 (2006)). Following this dosage regime, patients were stableor slightly improved for clinical outcomes including Expanded DisabilityStatus Scale (EDSS) score, ambulation index (AI), and nine-hole peg test(9-HPT). The most common adverse effects were gastrointestinal symptomsand flushing, and all adverse effects were reported as mild andreversible.

In an exemplary treatment regimen of fumaric acid esters for treatmentof psoriasis includes a gradual increase in dosage according to theschedule depicted in Table 3.

TABLE 3 Dosage schedule of fumaric acid esters used for patients withpsoriasis (Reproduced from Roll, et al., Indian J. Dermatol. Venereol.Leprol., 73: 133-7 (2007)). Week Fumaderm ® initial Fumaderm ® Dosage ofDMF 1 1-0-0  30 mg 2 1-0-1  60 mg 3 1-1-1  90 mg 4 1-0-0 120 mg 5 1-0-1240 mg 6 1-1-1 360 mg 7 2-1-1 480 mg 8 2-1-2 600 mg 9 2-2-2 720 mg

This schedule was shown to improve gastrointestinal tolerance (Nast A,et al., J. German Soc. Dermatol., 4:51-5 (2006)). Most patients treatedwith fumaric acids require two to four tablets of FUMADERM, fortreatment of psoriasis.

Therefore, daily dosages for fumaric acid esters can range from about 1mg to about 5,000 mg, preferably about 10 mg to about 2,500 grams, morepreferably about 50 mg to about 2,000 grams of a fumaric acid ester, ora pharmacologically active salt, derivative, analogue or prodrugthereof.

In some embodiments the compositions include DMF, MMF, or a combinationthereof. For DMF or MMF, the therapeutically effective amount can rangefrom about 1 mg/kg to about 50 mg/kg (e.g., from about 2.5 mg/kg toabout 20 mg/kg or from about 2.5 mg/kg to about 15 mg/kg). Effectivedoses will also vary, as recognized by those skilled in the art,dependent on route of administration, excipient usage, and thepossibility of co-usage with other therapeutic treatments including useof other therapeutic agents. For example, an effective dose of DMF orMMF to be administered to a subject, for example orally, can be fromabout 0.1 g to about 1 g or more than 1 g per day; from about 200 mg toabout 800 mg per day; from about 240 mg to about 720 mg per day; fromabout 480 mg to about 720 mg per day; or about 720 mg per day. The dailydose can be administered in separate administrations of 2, 3, 4, or 6equal doses.

In some embodiments of the one or more fumaric acid esters, orpharmacologically active salts, derivatives, analogues or prodrugsthereof are present in a pharmaceutical preparation. In some embodimentsthe composition is administered to the patient three times per day(TID). In some embodiments the pharmaceutical preparation isadministered to the patient two times per day (BID). In someembodiments, the composition is administered at least one hour before orafter food is consumed by the patient.

In some embodiments, the composition is administered as part of a dosingregimen. For example, the patient can be administered a first dose ofthe composition for a first dosing period; and a second dose of thecomposition for a second dosing period, optionally followed by one ormore additional doses for one or more additional dosing periods. Thefirst dosing period can be less than one week, one week, or more thanone week.

In some embodiments the dosage regime is a dose escalating dosageregime. The first dose can be a low dose. For example, in someembodiments, the composition includes DMF, and a low dose of DMF, forexample about 30 mg, can be the starting dose for a dose-escalationprotocol. Dose escalation can be continued until a satisfactorybiochemical or clinical response is reached. Next, the dosages can bemaintained or steadily reduced to a maintenance dose. In someembodiments, the final dosage can be about 1-2 grams per day (i.e., 6tablets of FUMADERM).

Studies on the use of fumaric acid esters to treat psoriasis show thatdosage may not be related to body weight or to the activity of thedisease (Nast A, et al., J. German Soc. Dermatol., 4:51-5 (2006)).Accordingly, the dosage and dosage regime for each patient can beadjusted according to the individual's response and the onset orseverity of adverse effects.

The most common side effects are gastrointestinal symptoms such asabdominal pain, diarrhea, nausea and malaise. These signs and symptomsoccur primarily within the first few weeks after initiation of treatmentand within 90 minutes to six hours after oral intake of the drug. Theylast for several minutes up to half an hour and can be alleviated byintake of tablets with milk.

Flushing of the skin is another common complaint, ranging from rapidsensation of heat to long-lasting facial redness. Improvement of thelatter side effect has been seen on treatment with acetylsalicylic acidbut this has not yet been confirmed scientifically. Typically, theadverse effects discussed above are dose-dependent and they decrease infrequency during the course of the treatment.

Less commonly observed side effects are lymphocytopenia, leukocytopeniaand elevated eosinophil counts. A decrease of lymphocytes below 500/mm³should lead to dosage reduction or withdrawal of treatment. Theeosinophilia is transient and usually observed between the fourth andtenth week of treatment.

Rarely, moderate elevations of liver enzymes and bilirubin have beenobserved. Proteinuria has been noted too, but it proved to be transient.An increased risk for infections has not been documented.

Relapse or rebound phenomena do not typically occur using fumaric acidesters such as FUMADERM. Therefore, treatment may be discontinuedabruptly if needed.

The current labeled dosing of hydroxyurea for sickle cell disease callsfor the administration of an initial dose of 15 mg/kg/day in the form ofa single dose, with monitoring of the patient's blood count every 2weeks. If the blood counts are in an acceptable range, the dose may beincreased by 5 mg/kg/day every 12 weeks until the MTD of 35 mg/kg/day isreached. Pharmaceutical compositions can contain 1 mg/kg to 50 mg/kg offumaric acid ester, preferably MMF, in combination with 1 mg/kg to 35mg/kg of HU. The combination formulation can contain 5, 10, 15, 20, 25,30, 35, 40, 45 or 50 mg/kg of HU.

D. Formulations

Pharmaceutical compositions including a fumaric acid ester, orpharmacologically active salt, derivative, analogue, or prodrug thereofare disclosed. The pharmaceutical compositions may be for administrationby oral, parenteral (intramuscular, intraperitoneal, intravenous (IV) orsubcutaneous injection), transdermal (either passively or usingiontophoresis or electroporation), or transmucosal (nasal, vaginal,rectal, or sublingual) routes of administration or using bioerodibleinserts and can be formulated in unit dosage forms appropriate for eachroute of administration.

Red blood cells, which are cells of erythroid lineage, are the primaryproducers of hemoglobin. Therefore, in a preferred embodiment thefumaric acid esters are administered to a subject in an effective amountto induce HbF in hematopoietic stems cells. In the early fetus,erythropoiesis takes place in the mesodermal cells of the yolk sac. Bythe third or fourth month, erythropoiesis moves to the spleen and liver.After seven months, erythropoiesis occurs primarily in the bone marrow,however, in certain disease states erythropoiesis can also occursoutside the bone marrow, within the spleen or liver, in adults.Therefore, in some embodiments, the compositions are administered in aneffective amount to induce HbF expression in cells of erythroid lineagein the bone marrow (i.e., the red bone marrow), the liver, the spleen,or combinations thereof.

Preferably the composition induces HbF in cells synthesizing orcommitted to synthesize hemoglobin. For example, in preferredembodiments, the fumaric acid ester, or pharmacologically active salt,derivative, analogue, or prodrug thereof induces HbF in basophilicnormoblast/early normoblast also commonly called erythroblast,polychromatophilic normoblast/intermediate normoblast, orthochromaticnormoblast/late normoblast, or a combination thereof.

In a preferred embodiment, the composition is an oral formulation. Oralformulations of DMF or MMF such as FUMADERM can be absorbed by the smallintestine where MMF can enter systemic circulation.

In some embodiments, the composition is administered locally, to thesite in need of therapy. Although red blood cells are the primaryproducers of hemoglobin, reports indicate that other, non-hematopoieticcells, including macrophage, retinal pigment cells, and alveolarepithelial cells such as alveolar type II (ATII) cells and Clara cellswhich are the primary producers of pulmonary surfactant, also synthesizehemoglobin (Newton, et al., J. Biol. Chem., 281(9)5668-5676 (2006),Tezel, et al., Invest. Ophthalmol. Vis. Sci., 50(4):1911-9 (2009), Liu,et al., Proc. Natl. Acad. Sci. USA, 96(12)6643-6647 (1999)). Thesefindings are consistent with the conclusion that the expression ofhemoglobin by non-erythroid cells at interfaces where oxygen-carbondioxide diffusion occurs may be an adaptive mechanism to facilitateoxygen transport.

Therefore, in some embodiments, the composition is administered locallyto interfaces where oxygen-carbon dioxide diffusion occurs, includingbut not limited, to the eye or lungs.

In some embodiments, the composition is administered locally to the eyeto treat a retinopathy, or another ocular manifestation associated withsickle cell disease, or a related disorder.

1. Formulations for Enteral Administration

In a preferred embodiment the compositions are formulated for oraldelivery. Oral solid dosage forms are described generally in Remington'sPharmaceutical Sciences, 18^(th) Ed. 1990 (Mack Publishing Co. EastonPa. 18042) at Chapter 89. Solid dosage forms include tablets, capsules,pills, troches or lozenges, cachets, pellets, powders, or granules orincorporation of the material into particulate preparations of polymericcompounds such as polylactic acid, polyglycolic acid, etc., or intoliposomes. Such compositions may influence the physical state,stability, rate of in vivo release, and rate of in vivo clearance of thedisclosed. See, e.g., Remington's Pharmaceutical Sciences, 18^(th) Ed.(1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712 which areherein incorporated by reference. The compositions may be prepared inliquid form, or may be in dried powder (e.g., lyophilized) form.Liposomal or proteinoid encapsulation may be used to formulate thecompositions. Liposomal encapsulation may be used and the liposomes maybe derivatized with various polymers (e.g., U.S. Pat. No. 5,013,556).See also, Marshall, K. In: Modern Pharmaceutics Edited by G. S. Bankerand C. T. Rhodes Chapter 10, 1979. In general, the formulation willinclude the peptide (or chemically modified forms thereof) and inertingredients which protect peptide in the stomach environment, andrelease of the biologically active material in the intestine.

The fumaric acid ester, or pharmacologically active salt, derivative,analogue, or prodrug thereof may be chemically modified so that oraldelivery of the compound is efficacious. Generally, the chemicalmodification contemplated is the attachment of at least one moiety tothe component molecule itself, where the moiety permits uptake into theblood stream from the stomach or intestine, or uptake directly into theintestinal mucosa. Also desired is the increase in overall stability ofthe component or components and increase in circulation time in thebody. PEGylation is a preferred chemical modification for pharmaceuticalusage. Other moieties that may be used include: propylene glycol,copolymers of ethylene glycol and propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone,polyproline, poly-1,3-dioxolane and poly-1,3,6-tioxocane [see, e.g.,Abuchowski and Davis (1981) “Soluble Polymer-Enzyme Adducts,” in Enzymesas Drugs. Hocenberg and Roberts, eds. (Wiley-Interscience: New York,N.Y.) pp. 367-383; and Newmark, et al. (1982) J. Appl. Biochem.4:185-189].

Another embodiment provides liquid dosage forms for oral administration,including pharmaceutically acceptable emulsions, solutions, suspensions,and syrups, which may contain other components including inert diluents;adjuvants such as wetting agents, emulsifying and suspending agents; andsweetening, flavoring, and perfuming agents.

Controlled release oral formulations may be desirable. Fumaric acidesters, or pharmacologically active salt, derivatives, analogues, orprodrugs thereof can be incorporated into an inert matrix which permitsrelease by either diffusion or leaching mechanisms, e.g., gums. Slowlydegenerating matrices may also be incorporated into the formulation.Another form of a controlled release is based on the Oros therapeuticsystem (Alza Corp.), i.e., the drug is enclosed in a semipermeablemembrane which allows water to enter and push drug out through a singlesmall opening due to osmotic effects.

For oral formulations, the location of release may be the stomach, thesmall intestine (the duodenum, the jejunem, or the ileum), or the largeintestine. Preferably, the release will avoid the deleterious effects ofthe stomach environment, either by protection of the agent (orderivative) or by release of the agent (or derivative) beyond thestomach environment, such as in the intestine. To ensure full gastricresistance a coating impermeable to at least pH 5.0 is essential.Examples of the more common inert ingredients that are used as entericcoatings are cellulose acetate trimellitate (CAT),hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55,polyvinyl acetate phthalate (PVAP), Eudragit L30D™, Aquateric™,cellulose acetate phthalate (CAP), Eudragit L™, Eudragit S™, andShellac™. These coatings may be used as mixed films.

2. Topical or Mucosal Delivery Formulations

Compositions can be applied topically. The compositions can be deliveredto the lungs while inhaling and traverses across the lung epitheliallining to the blood stream when delivered either as an aerosol or spraydried particles having an aerodynamic diameter of less than about 5microns.

A wide range of mechanical devices designed for pulmonary delivery oftherapeutic products can be used, including but not limited to,nebulizers, metered dose inhalers, and powder inhalers, all of which arefamiliar to those skilled in the art. Some specific examples ofcommercially available devices are the Ultravent™ nebulizer(Mallinckrodt Inc., St. Louis, Mo.); the Acorn II™ nebulizer (MarquestMedical Products, Englewood, Colo.); the Ventolin™ metered dose inhaler(Glaxo Inc., Research Triangle Park, N.C.); and the Spinhaler™ powderinhaler (Fisons Corp., Bedford, Mass.).

Formulations for administration to the mucosa will typically be spraydried drug particles, which may be incorporated into a tablet, gel,capsule, suspension or emulsion. Standard pharmaceutical excipients areavailable from any formulator. Oral formulations may be in the form ofchewing gum, gel strips, tablets or lozenges.

Transdermal formulations may also be prepared. These will typically beointments, lotions, sprays, or patches, all of which can be preparedusing standard technology. Transdermal formulations will require theinclusion of penetration enhancers.

3. Controlled Delivery Polymeric Matrices

Controlled release polymeric devices can be made for long term releasesystemically following implantation of a polymeric device (rod,cylinder, film, disk) or injection (microparticles). The matrix can bein the form of microparticles such as microspheres, where peptides aredispersed within a solid polymeric matrix or microcapsules, where thecore is of a different material than the polymeric shell, and thepeptide is dispersed or suspended in the core, which may be liquid orsolid in nature. Unless specifically defined herein, microparticles,microspheres, and microcapsules are used interchangeably. Alternatively,the polymer may be cast as a thin slab or film, ranging from nanometersto four centimeters, a powder produced by grinding or other standardtechniques, or even a gel such as a hydrogel.

Either non-biodegradable or biodegradable matrices can be used fordelivery of disclosed compounds, although biodegradable matrices arepreferred. These may be natural or synthetic polymers, althoughsynthetic polymers are preferred due to the better characterization ofdegradation and release profiles.

The polymer is selected based on the period over which release isdesired. In some cases linear release may be most useful, although inothers a pulse release or “bulk release” may provide more effectiveresults. The polymer may be in the form of a hydrogel (typically inabsorbing up to about 90% by weight of water), and can optionally becrosslinked with multivalent ions or polymers.

The matrices can be formed by solvent evaporation, spray drying, solventextraction and other methods known to those skilled in the art.Bioerodible microspheres can be prepared using any of the methodsdeveloped for making microspheres for drug delivery, for example, asdescribed by Mathiowitz and Langer, J. Controlled Release 5:13-22(1987); Mathiowitz, et al., Reactive Polymers 6:275-283 (1987); andMathiowitz, et al., J. Appl. Polymer Sci. 35:755-774 (1988).

The devices can be formulated for local release to treat the area ofimplantation or injection—which will typically deliver a dosage that ismuch less than the dosage for treatment of an entire body—or systemicdelivery. These can be implanted or injected subcutaneously, into themuscle, fat, or swallowed.

IV. Methods of Diagnosis

The methods of treatment disclosed herein can include a first step ofselecting a subject for treatment. In some embodiments, the subject isselected for treatment when the subject exhibits one or more of theclinical symptoms of sickle cell disease, beta-thalassemia, or a relateddisorder such as those discussed above. In some embodiments, the subjectis selected for treatment when the subject exhibits a genetic orbiochemical indicator of sickle cell disease, beta-thalassemia, or arelated disorder. For example, the subject can be selected for treatmentbased on identification of a genetic alteration, defect, or mutation inthe beta-globin gene or an expression control sequence thereof, bybiochemical or morphological alterations in hemoglobin or hemoglobinsynthesizing cells, or combinations thereof.

In some embodiments, the subject is selected when a combination ofclinical symptoms and genetic or biochemical alterations are identified.In some embodiments, the subject is selected based on one or moreclinical symptoms, or one or more genetic or biochemical alterations.For example, subjects can be selected for treatment based on theidentification of a genetic alteration, a biochemical or morphologicalalteration, or a combination thereof, before the subject exhibitsclinical symptoms of sickle cell disease, beta-thalassemia, or a relateddisorder.

A. Identification of Genetic Alterations

In some embodiments, the subject is selected for treatment based onidentification of one or more genetic alterations in one or more allelesof the human beta-globin gene or expression control sequence thereof.Genetic alterations indicative of sickle cell disease, beta-thalassemia,or related disorders include the exemplary mutations discussed above, orother mutations that lead to a reduction in the synthesis, structure, orfunction of human beta-globin protein.

Methods of selecting a subject having one or more genetic alterations inone or more alleles of the beta-globin gene or expression controlsequences thereof include the steps of obtaining a biological samplecontaining nucleic acid from the subject and detecting the presence orabsence one or more genetic alterations in one or more alleles of thebeta-globin gene or expression control sequences thereof in thebiological sample. Any biological sample that contains the DNA of thesubject to be diagnosed can be employed, including tissue samples andblood samples, with nucleated blood cells being a particularlyconvenient source. The DNA may be isolated from the biological sampleprior to testing the DNA for the presence or absence of the geneticalterations.

The detecting step can include determining whether the subject isheterozygous or homozygous for a genetic alteration. The step ofdetecting the presence or absence of the genetic alteration can includethe step of detecting the presence or absence of the alteration in bothchromosomes of the subject (i.e., detecting the presence or absence ofone or two alleles containing the marker or functional polymorphism).More than one copy of a genetic alterations (i.e., subjects homozygousfor the genetic marker) can indicate a greater risk of developing sicklecell disease, beta-thalassemia, or related disorder. In someembodiments, the subject is heterozygous for two or more geneticalterations in the beta-globin gene (also referred to herein as doubleheterozygotes, triple heterozygotes, etc.). One copy of two or moregenetic alterations in the beta-globin gene can indicate a greater riskof developing sickle cell disease, beta-thalassemia, or relateddisorder.

The process of determining the genetic sequence of human beta-globingene is referred to as genotyping. In some embodiments, the humanbeta-globin gene is sequenced. Methods for amplifying DNA fragments andsequencing them are well known in the art. For example, automatedsequencing procedures that can be utilized to sequence the beta-globingene, include, but not limited to, sequencing by mass spectrometrysingle-molecule real-time sequencing (Pacific Bio), ion semiconductor(ion torrent sequencing), pyrosequencing (454), sequencing by synthesis(Illumina), sequencing by ligation (SOLiD sequencing), chain termination(Sanger sequencing).

In some embodiments, the genotype of the subject is determined byidentifying the presence of one or more single nucleotide polymorphisms(SNP) associated with sickle cell disease, beta-thalassemia, or arelated disorder. Methods for SNP genotyping are generally known in theart (Chen et al., Pharmacogenomics J., 3(2):77-96 (2003); Kwok, et al.,Curr. Issues Mol. Biol., 5(2):43-60 (2003); Shi, Am. J.Pharmacogenomics, 2(3):197-205 (2002); and Kwok, Annu. Rev. GenomicsHum. Genet., 2:235-58 (2001)).

SNP genotyping can include the steps of collecting a biological samplefrom a subject (e.g., sample of tissues, cells, fluids, secretions,etc.), isolating genomic DNA from the cells of the sample, contactingthe nucleic acids with one or more primers which specifically hybridizeto a region of the isolated nucleic acid containing a target SNP underconditions such that hybridization and amplification of the targetnucleic acid region occurs, and determining the nucleotide present atthe SNP position of interest, or, in some assays, detecting the presenceor absence of an amplification product (assays can be designed so thathybridization and/or amplification will only occur if a particular SNPallele is present or absent). In some assays, the size of theamplification product is detected and compared to the length of acontrol sample; for example, deletions and insertions can be detected bya change in size of the amplified product compared to a normal genotype.

The neighboring sequence can be used to design SNP detection reagentssuch as oligonucleotide probes and primers. Common SNP genotypingmethods include, but are not limited to, TaqMan assays, molecular beaconassays, nucleic acid arrays, allele-specific primer extension,allele-specific PCR, arrayed primer extension, homogeneous primerextension assays, primer extension with detection by mass spectrometry,pyrosequencing, multiplex primer extension sorted on genetic arrays,ligation with rolling circle amplification, homogeneous ligation,multiplex ligation reaction sorted on genetic arrays,restriction-fragment length polymorphism, single base extension-tagassays, and the Invader assay. Such methods may be used in combinationwith detection mechanisms such as, for example, luminescence orchemiluminescence detection, fluorescence detection, time-resolvedfluorescence detection, fluorescence resonance energy transfer,fluorescence polarization, mass spectrometry, and electrical detection.

Other suitable methods for detecting polymorphisms include methods inwhich protection from cleavage agents is used to detect mismatched basesin RNA/RNA or RNA/DNA duplexes (Myers et al., Science, 230:1242 (1985);Cotton, et al., PNAS, 85:4397 (1988); and Saleeba, et al., Meth.Enzymol., 217:286-295 (1992)), comparison of the electrophoreticmobility of variant and wild type nucleic acid molecules (Orita et al.,PNAS, 86:2766 (1989); Cotton, et al, Mutat. Res., 285:125-144 (1993);and Hayashi, et al., Genet. Anal. Tech. Appl., 9:73-79 (1992)), andassaying the movement of polymorphic or wild-type fragments inpolyacrylamide gels containing a gradient of denaturant using denaturinggradient gel electrophoresis (DGGE) (Myers et al., Nature, 313:495(1985)). Sequence variations at specific locations can also be assessedby nuclease protection assays such as Rnase and 51 protection orchemical cleavage methods.

Another method for genotyping SNPs is the use of two oligonucleotideprobes in an oligonucleotide ligation assay (OLA) (U.S. Pat. No.4,988,617). In this method, one probe hybridizes to a segment of atarget nucleic acid with its 3′-most end aligned with the SNP site. Asecond probe hybridizes to an adjacent segment of the target nucleicacid molecule directly 3′ to the first probe. The two juxtaposed probeshybridize to the target nucleic acid molecule, and are ligated in thepresence of a linking agent such as a ligase if there is perfectcomplementarity between the 3′-most nucleotide of the first probe withthe SNP site. If there is a mismatch, ligation would not occur. Afterthe reaction, the ligated probes are separated from the target nucleicacid molecule, and detected as indicators of the presence of a SNP.

Other methods that can be used to genotype the SNPs includesingle-strand conformational polymorphism (SSCP), and denaturinggradient gel electrophoresis (DGGE). SSCP identifies base differences byalteration in electrophoretic migration of single stranded PCR products.Single-stranded PCR products can be generated by heating or otherwisedenaturing double stranded PCR products. Single-stranded nucleic acidsmay refold or form secondary structures that are partially dependent onthe base sequence. The different electrophoretic mobilities ofsingle-stranded amplification products are related to base-sequencedifferences at SNP positions. DGGE differentiates SNP alleles based onthe different sequence-dependent stabilities and melting propertiesinherent in polymorphic DNA and the corresponding differences inelectrophoretic migration patterns in a denaturing gradient gel.

Sequence-specific ribozymes (U.S. Pat. No. 5,498,531) can also be usedto score SNPs based on the development or loss of a ribozyme cleavagesite. Perfectly matched sequences can be distinguished from mismatchedsequences by nuclease cleavage digestion assays or by differences inmelting temperature. If the SNP affects a restriction enzyme cleavagesite, the SNP can be identified by alterations in restriction enzymedigestion patterns, and the corresponding changes in nucleic acidfragment lengths determined by gel electrophoresis.

B. Identification of Biochemical and Morphological Alterations

In some embodiments, subjects are selected for treatment based onidentification of biochemical or morphological alterations orabnormalities in hemoglobin, or hemoglobin synthesizing cells such ashematopoietic stem cells, erythrocyte progenitor cells, erythrocytes,macrophage, retinal pigment epithelial cells, alveolar type II (ATII)cells, and others. The methods typically include identifying one or morebiochemical or morphological alterations that is associated with agenetic alteration in the human beta-globin gene, or otherwisediagnostic of sickle cell disease, a beta-thalassemia, or a relateddisorder. Methods of diagnosing sickle cell disease, beta-thalassemia,or a related disorder according to biochemical or morphologicalalterations in the hemoglobin or hemoglobin synthesizing cells are knownin the art, and include but are not limited to, analysis of erythrocytemorphology, osmotic fragility, hemoglobin composition, globin synthetsisrates, and red blood cell indices (Rowley, American Journal ofHematology, 1(1):129-137, (1976)).

In some embodiments, the method includes testing a subject's blood forHbS, and selecting the subject for treatment if HbS is present. Methodsfor testing a subject's blood for the presence of HbS include solubilitytests (e.g., SICKLEDEX) and sickling test. The SICKLEDEX test operateson the principle that Hb-S tends to form tactoids or liquid crystalswithin the erythrocytes under conditions of low oxygen tension resultingin the characteristic “sickle shape” distortion of the red cell. Areducing agent (i.e., dithionite) is mixed with whole blood and buffer.If Hb-S is present, it becomes insoluble and forms a cloudy suspension.Other hemoglobins are more soluble and will form a transparent solution.A sickling test can be used to determine if a red blood cell changesinto a sickle shape after a blood sample is mixed with a reducing agentand identifying morphological changes to shape of red blood cells (i.e.,“sickling”) by microscopy.

Other suitable tests include, hemoglobin electrophoresis, which employsgel electrophoretic techniques to separate out the various types ofhemoglobin from a blood sample obtained from the subject. The test candetect abnormal levels of HbS, as well as other abnormal hemoglobins,such as hemoglobin C. It can also be used to determine whether there isa deficiency of any normal form of hemoglobin, as in variousthalassemias. Alternatives to electrophoretic techniques includeisoelectric focusing and chromatographic techniques.

Other tests that can be used to select a subject for treatment with thecompositions and methods disclosed herein include tests typicallyemployed as part of a hemoglobinopathy screen, for example, a completeblood count (CBC) or iron study (ferritin). For example, a blood countcan be used to detect anemia, and a blood smear and be used to identifysickled cells.

EXAMPLES Example 1 Monomethylfumarate (MMF) Induces γ-Globin (Hbf) Geneand Protein Expression in Cells of Erythroid Lineage Materials andMethods

Pharmaceutical Agents

The fumaric acid esters dimethylfumarate (DMF) and monomethylfumarate(MMF) are the primary constituents of Fumaderm and BG00012, drugscurrently marketed for treatment of psoriasis. BG00012 is alsocompleting phase III clinical testing for treatment of multiplesclerosis. MMF is the major bioactive component of each.

Cell Culture

KU812, a human leukemic cell line that expresses the fetal γ-globin andadult β-globin genes, is a commonly used system for screening anddiscovery of novel HbF inducers; this is because of comparable globingene response patterns in KU812 and primary erythroid cells aftertreatments with drug inducers.

Results

KU812 cells were cultured in the presence or absence of MMF for timeperiods ranging from 0-24 hours and evaluated for changes in γ-globingene expression relative to 18S ribosomal RNA expression (internalexperimental control) by qPCR (FIG. 1). In comparison to control,untreated (UT) cells, significant increases in γ-globin gene expressionwere observed as early as 3 hours post-incubation with low-dose (100 μM)MMF treatment. This MMF-induced increase in γ-globin expressionpersisted up to 24 h post-exposure to the compound. Data in FIG. 1 arerepresented as means±standard error of the mean (SEM); *P<0.05, **P<0.01and ***P<0.001.

FIG. 1 shows that 100 μM MMF increases the expression of γ-globin geneexpression in KU812 cells ˜2-4-fold depending upon the incubation time.FIG. 2 shows the induction of γ-globin transcription by known HbFinducers in KU812 cells cultured under conditions similar to thosedescribed above. FIG. 2 is reproduced from MaKala et al., Anemia, Volume2012, Article ID 428137) (2012). The last bar of FIG. 2 shows data forhydroxyurea (HU). At 100 μM, the same concentration that we used forMMF, HU induces γ-globin gene expression ˜2-fold. This data supports theconclusion that MMF is at least equivalent, or even better than HU, interms of its ability to induce γ-globin gene expression in this cellsystem.

Example 2 Monomethylfumarate (MMF) Drives Expression by Activation theγ-Globin (Hbf) Gene Promoter Materials and Methods

-   From MaKala et al., Anemia, Volume 2012, Article ID 428137) (2012)

KU812 Stable Lines

KU812 stable cell lines were created by co-transfecting wild-type KU812cells with pEGFP-NI (G418 selectable marker) and the μLCRβprRlucAγprFluc dualreporter a kind gifts from Dr. George Stamatoyannopoulos(University of Washington). Briefly, the 315-bp human β-globin genepromoter sequence was inserted upstream of the Renilla along with apolyadenylation signal downstream to create PβprRluc. Likewise, 1.4 kbof human Aγ-globin promoter was inserted upstream of firefly luciferaseto create AγprFluc. The μLCR (locus control region), PβprRluc, andAγprFluc fragments were subsequently cloned into the mammalian vector,pRL-null. The dual-luciferase reporter lines were produced using 10 μgeach of linearized μLCRβprRluc AγprFluc and pEGFP-NI plasmidsco-transfected into KU812 cells by electroporation at 260 V, 975 μF(Bio-Rad, Hercules Calif.). After 72 hr, G418 was added at aconcentration of 900 μg/μl for 3 days then maintained under selectionpressure indefinitely at a concentration of 400 μg/μl. KU812 stablelines were treated with the various drugs at the same concentrationsdescribed above. FK228 and analogues were screened at concentrationsbetween 1-1000 nM for 48 hr and cell toxicity was monitored by 2% Trypanblue exclusion. The effect of drug treatments on γ-globin and β-globinpromoter activity was monitored by luciferase assay.

Dual Luciferase Assay

Luciferase activity was monitored under the different experimentalconditions using the Dual Luciferase Assay Reporter System (Promega,Madison, Wis.). The activity of firefly luciferase represents γ-globinpromoter activity (γF), while the renilla luciferase is the read-out forβ-globin promoter activity (βR). The β-globin promoter was strategicallycloned between the LCR and γ-globin promoter to increase β expression,while simultaneously increasing the sensitivity of detection of γ-globingene inducers. After drug treatments, KU812 stable cells were washedwith 1× phosphate buffered saline and lysed in 1× Passive Lysis Bufferfor 15 min, then protein extracts were added to the Luciferase AssayReagent II and firefly luciferase activity quantified in a TurnerDesigns TD-20/20 luminometer (Sunnyvale, Calif.). Tomeasure βR activity,Stop & Glo Reagents was added to measure the renilla luciferaseactivity. Total protein was determined by Bradford assay on a Beckman DU640 spectrophotometer (Chaska, Minn.) and luciferase activity wascorrected for total protein.

Results

The fumaric acid ester was also in the KU812 dual-luciferase reportersystem, an assay system using a stable KU812 cell line created with theμLCRβprRlucγprFluc construct containing a 3.1-kb μLCR cassette linked toa 315-bp human β-globin promoter driving the renilla and a 1.4-kbAγ-globin promoter driving the firefly luciferase genes. Since, thefirefly luciferase gene (γF) has approximately 50% greater luminescencethan the renilla gene (βR), renilla activity was multiplied by two toadjust for the difference in luminescence yielding the γ/γ+2β finalmeasurement. This assay system was previously reported as an efficaciousscreening tool for the identification of γ-globin gene activators(MaKala et al., Anemia, Volume 2012, Article ID 428137) (2012)).

Luciferase activity, an indicator of γ-globin promoter induction, wasmonitored KU812 μLCRβprRlucγprFluc-stable cells cultured in the presenceor absence (untreated control, UT) of MMF at varying dosages for 48 houraccording to the method discussed above and in MaKala et al., Anemia,Volume 2012, Article ID 428137) (2012). MMF induced γ-globin promoteractivity in a dose-dependent manner (FIG. 3). Cell viability by Trypanblue exclusion remained at 90-95% for the concentrations shown,indicating that even at higher concentrations, MMF exhibits little or nocellular toxicity.

Example 3 Dimethylfumarate (DMF) Drives Expression by Activation theγ-Globin (Hbf) Gene Promoter

MMF is the primary bioactive metabolite derived from metabolism ofFUMADERM and BG00012, the fumaric acid ester drugs presently usedclinically, and DMF is the primary ingredient. Therefore, theeffectiveness of DMF was tested as an inducer of γ-globin geneexpression under experimental conditions identical to those describedabove in Example 2. As with MMF, treatment of cells with DMF alsoinduced γ-globin promoter activity significantly (FIG. 4). Atconcentrations in the 100-200 μM range, induction of γ-globin promoteractivity increased ˜2-10 fold in comparison to control, untreated (UT)cells. However, at higher concentrations the effectiveness of DMF as aninducer of γ-globin promoter activity declined substantially. Trypanblue exclusion revealed that this decrease was likely due to an increasein cellular toxicity as indicated by a decrease in cell viability.

Example 4 Monomethylfumarate (MMF) Induces γ-Globin (Hbf) Gene andProtein Expression in a Retinal Pigment Epithelial (RPE) Cell Line

Red blood cells, cells of erythroid lineage, are the primary producersof hemoglobin. However, recent reports suggest that other,non-hematopoietic cells are capable of the same. This includes retinalpigment epithelial (RPE) cells, a cell type critical to normal visualfunction. Therefore, an assay was designed to test the induction ofγ-globin gene expression by MMF in ARPE-19, a transformed human RPE cellline commonly used as an in vitro model of human RPE ARPE-19 cells werecultured in the presence or absence or MMF (100 μM) for varying periodsof time (0-24 h) and the expression of γ-globin analyzed by reversetranscriptase-polymerase chain reaction (RT-PCR) (FIG. 5A). Atime-dependent increase in γ-globin gene expression was observed inthese cells when cultured in the presence of MMF.

The results presented in FIG. 5A were data were corroborated usingreal-time quantitative PCR (qPCR) (FIG. 5B). Data are represented asmean±SEM; *P<0.05, **P<0.01.

Immunofluorescence localization techniques utilizing a FITC-labeledanti-HbF antibody confirmed that the MMF-induced increase in γ-globinmRNA was associated with a corresponding increase in HbF proteinexpression.

Example 5 Monomethylfumarate (MMF) Induces γ-Globin (Hbf) Gene andProtein Expression in Primary Retinal Pigment Epithelial (RPE) Cells

The effect of MMF on primary RPE cells was also investigated. RPE cellswere isolated from the eyes of humanized mice and used to establishprimary RPE cell cultures. These animals have been geneticallyengineered such that they express human rather than mouse beta and gammaglobin genes and hence synthesize human hemoglobin. Primary RPE cellswere cultured in the presence or absence of MMF at concentrationsranging from 0-1000 μM for a period of 9 hours. Total RNA was preparedand γ-globin gene expression analyzed by qPCR. A dose-dependent increasein γ-globin gene expression was observed also in these cells whencultured in the presence of MMF (0-1000 μM) (FIG. 6). Data arerepresented as mean±SEM; *P<0.01, **P<0.001.

Example 6 Evaluation of the Induction of γ-Globin by DMF and MMF Methodsand Materials

Induction of γ-globin by DMF and MMF (Sigma, St. Louis, Mo.) in thedual-luciferase KU812 stable line using a dual luciferase assay wasinvestigated. HU (100 μM; Sigma) was included as a positive control andcell viability was monitored by trypan blue exclusion. Findings in KU812cells were confirmed in human primary erythroid progenitor cells grownin liquid culture using a published protocol. Globin expression wasmeasured by qPCR also as previously published. HbF protein was measuredrelative to that of isotype control using FITC conjugated anti-human HbFantibody (1:1000; Santa Cruz Biotechnology, Santa Cruz, Calif.) andfluorescence activated cell sorting (see supplemental methods fordetails). HbF protein expression was confirmed by Western blot analysisusing anti-human HbF antibody (1:1000; Bethyl Laboratories, Inc.,Montgomery, Tex.) and horseradish peroxidase-conjugated sheep IgG(1:1000; Santa Cruz).

Identical experiments were performed using the human RPE cell lineARPE-19, an established model for the study of RPE19, and primary RPEcell cultures established from the eyes of HbAA- and HbSS-expressingTownes humanized knock-in SCD mice (Jackson Laboratories, Bar Harbor,Me.) per our published method. Additionally, MMF (1 mM finalconcentration) or PBS (control) was injected intravitreally into theeyes of HbAA and HbSS mice and retinal γ-globin and HbF expressionanalyzed by qPCR and immunofluorescence 24 h post-injection. Animalstudies were approved by the Georgia Regents University InstitutionalCommittee for Animal Use in Research and Education.

Primary Erythroid Culture

Erythroid progenitors were generated in vitro from adult CD34^(|) stemcells (STEMCELL Technologies, Inc. Vancouver, Canada) using a 2-stageculture system that achieves terminal erythroid differentiation's. CD34⁺stem cells (500,000) were grown in First medium consisting of IscoveModified Dulbecco Media containing human AB serum, interleukin-3 (10ng/mL), stem cell factor (10 ng/mL) and erythropoietin (2 IU/mL). On day7, the erythroblasts were placed in Second medium with 2 IU/mLerythropoietin for the duration. On day 8, erythroid cells were treatedwith monomethylfumarate (MMF, 1000 μM), dimethylfumarate (DMF, 200 μM)or hydroxyurea (HU, 100 μM). Then cells were harvested for total RNA andprotein for qPCR, FACS and western blot analyses.

Fluorescence Activated Cell Sorting (FACS)

After drug treatments, 500,000 cells were washed twice with phosphatebuffered saline and then fixed in 4% paraformaldehyde and permeated withice-cold acetone/methanol (4:1). Cells were incubated withanti-γ-globin-FITC antibody (Santa Cruz Biotechnology, Santa Cruz,Calif.) in PBT (PBS/01% BSA/0.1% triton X100) solution for 20 min tostain intracellular HbF antigens. The labeled cells were analyzed byBectin Dickerson LSR-II flow cytometer (BD Bioscience). All experimentswere performed in triplicate.

Intravitreal Injection

HbAA- and HbSS-expressing Townes humanized knock-in sickle cell diseasemice (6 weeks old; n=6) were used for intravitreal injection of MMFfollowing our published protocol₉. Briefly, animals were weighed andanesthetized using 17 μL (1 μL/g body weight) of a solution of ketamine(80 mg/mL) and xylazine (12 mg/mL). Then 5 μL of proparacaine solution(5% w/v) was administered topically to the eyes. MMF (1 μL; 10 mMsolution prepared in PBS) was then injected into the vitreous body ofthe right eye of each animal at the limbus; the left eye served as acontralateral control and received an equal volume of phosphate bufferedsaline (PBS, 0.01 M pH 7.4). Taking into account a total estimatedvitreous volume of 10 μL per mouse eye, the final concentration of MMFachieved in our experimental system was 1 mM. At 24 h postinjection,mice were sacrificed via CO₂ inhalation, and eyes were harvested. Someeyes (n=3 per treatment group) were flash frozen in liquid nitrogen andcryosectioned for use in immunofluorescence analyses while the remainingwere dissected to isolate RPE/eyecup from neural retina and total RNAprepared.

Results

Pharmacologic induction of HbF remains the best treatment approach toameliorate the clinical complications of SCD. The pleiotropic actions ofFAE in a broad spectrum of tissues, high tolerability and oralbioavailability, and recent FDA approval of Tecfidera (BG-12; BiogenIdec, Weston, Mass.) for use in multiple sclerosis make these agentsattractive for rapid extrapolation to clinical trials in SCD. Therefore,the ability of DMF and MMF to induce γ-globin expression and HbFproduction in erythroid cells was investigated. The induction ofγ-globin promoter activity by DMF and MMF was observed in KU812 cells bydual luciferase assay with maximal induction at 200 μM for DMF and 1000μM MMF (FIGS. 7A and 7B); findings were confirmed in primary humanerythroid cells (FIGS. 7C-7E). Levels of γ/β-globin mRNA were inducedsignificantly by both DMF and MMF (4- and 8-fold, respectively; FIG.7C). FACS demonstrated a 28- and 32-fold increase in HbF positive cellsin the presence of these compounds (FIGS. 7D(1)-7D(4) and 7E), which issignificantly higher than levels produced by HU (15-fold); see also FIG.9. HbF protein expression was confirmed by Western blot (data notshown).

HbF protein production in ARPE-19 cells exposed to MMF (1000 μM) for 24h was evaluated using a FITC-conjugated HbF antibody and fluorescencemicroscopy (data not shown). Cell nuclei were counterstained with DAPI.Treatments identical to those detailed above were performed usingprimary RPE cells isolated from the eyes of HbAA- or HbSS-expressingTownes humanized knock-in sickle cell disease mice and the expression ofγ-globin mRNA evaluated by qPCR using primer pairs specific to the humanγ-globin gene.

Data (mean±SEM) are from at least five data points generated from atleast three independent drug treatments. For in vivo studies, sixanimals were included per group and samples were run in duplicate.Paired student t-test was performed and a P<0.05 was consideredsignificant.

These data demonstrate the ability of DMF and MMF to induce HbFsynthesis in human erythroid progenitors and support further testing ina pre-clinical sickle cell mouse model.

Following oral intake, DMF is not detectable in plasma as it is rapidlyhydrolyzed and converted into MMF. Based on this information, only MMFfor studies with RPE cells were used (FIGS. 8A-8G). Findings in ARPE-19and primary RPE cells mirrored closely those obtained in primaryerythroid progenitors. This work confirms a single prior report ofβ-globin gene expression in RPE14 (FIGS. 8A and C) and demonstrates forthe first time the induction of γ-globin expression and HbF productionin these cells by MMF and HU (FIGS. 8B, 8D, 8E and FIG. 11). These dataare further supported by in vivo studies demonstrating the significantelevation of γ-globin mRNA and HbF protein in RPE/eyecup and neuralretina isolated from the eyes of HbAA and HbSS mice injectedintravitreally with MMF (FIGS. 8F and 8G).

Though SR is thought to be largely a vascular disease, there is clinicalevidence of early, non-vascular cell involvement, specifically ofphotoreceptor cell (PRC) dysfunction. PRCs, first order neurons in thevisual pathway, have a high oxygen demand. Given their isolation from avascular supply, they depend solely upon RPE cells for metabolicsupport. The synthesis of Hb by non-erythroid cells has been reported atother interfaces where O₂/CO₂ diffusion occurs; this may also be thecase in RPE cells. The physiological importance of Hb production in RPEis yet to be determined; it is possible that defects in RPE Hbexpression may contribute to retinal dysfunction and degeneration inSCD3.

Little is known regarding the impact of HbF-inducing therapies in theretina. A recent study by Estepp et al. demonstrated an inversecorrelation between SR and plasma HbF concentration. Follow-up studieson a larger scale are required to substantiate HbF-inducing therapies totreat SR. Such therapy may confer benefit in patients of HbSS and HbSCgenotypes, where the incidence of SR is highest. The present findingsthat FAE induce γ-globin expression and HbF production are new andsupport the possible re-purposing of BG-12 for treatment of SCD.Additionally, a new cellular target was identified for the therapeuticmanagement of SR, a factor of high clinical relevance given the 10%incidence of vision loss and blindness among SCD patients and the lackof effective strategies for prevention and treatment.

Example 7 MMF Induces Expression of SLC22A4 (aka OCTN1)

Immunofluorescence analysis of human OCTN1 expression revealed therobust expression of the transporter in human primary erythroid cellsgenerated in liquid culture from adult CD34⁺ stem cells.

KU812, a human leukemic cell line that expresses the fetal γ-globin andadult β-globin genes, is a commonly used system for screening anddiscovery of novel HbF inducers (see above). FIG. 7 shows the robustinduction of γ-globin mRNA and HbF production in these cells by MMF.FIG. 11 shows OCTN1 expression is also induced in these cells by MMFtreatment (MMF, 1000 μM, 16 h). Data are represented as mean±standarderror of the mean; *p<0.05.

Red blood cells, cells of erythroid lineage, are the primary producersof hemoglobin. However, recent reports suggest other, nonhematopoieticcells to be capable of doing the same. This includes retinal pigmentepithelial (RPE) cells, a cell type critical to normal visual function.FIG. 8 shows that induction of γ-globin gene expression and HbF proteinproduction by MMF occurs in RPE. FIG. 12 shows that MMF also inducesOCTN1 expression in these cells; HU alone had little to no effect onOCTN1 expression. Data are represented as mean±SEM; *p<0.05.

Example 8 MMF Induces OCTN1 mRNA and Protein Expression in Primary RPECells Isolated from HbAA- and HbSS-Expressing Mouse Retinas

Given that ARPE-19 is a transformed human RPE cell line, the findings inRPE cells isolated freshly from the living animals as such cells wasinvestigated to provide a more accurate representation of RPE cells intheir native environment. Additionally, the humanized knock-in SCD mousemodel (the Townes mouse), a rodent model engineered such that animalsexpress human α, β, and γ globin rather than the rodent globin genes,allows for the pre-clinical study of parameters highly reflective of thehuman condition. The effects of MMF on OCTN1 expression in HbAA andHbSS-expressing primary RPE cells from this model was investigated.Treatment with 1000 μM MMF induced expression of OCTN1 robustly both atthe RNA and protein level. (FIG. 13, *p<0.001).

Example 9 MMF Induces OCTN1 Protein Expression In Vivo in HbAA- andHbSS-Expressing Mouse Retinas

To determine whether the findings obtained in isolated retinal cells canbe extrapolated to the in vivo condition, MMF (1 mM final concentration)was delivered intravitreally into the eyes of HbAA- and HbSS-expressingmice. 24 h post-injection, animals were sacrificed and OCTN1 expressionevaluated by immunofluorescence. OCTN1 protein expression wasupregulated throughout the entire retina (FIG. 14). Given that theseanimals are of a pigmented background and RPE is loaded with melaninpigment, a property that may interfere or mask fluorescent signalintensity, the expression of OCTN1 in the RPE cell layer specifically isnot as apparent using this method. However, based upon data in primaryRPE cells isolated from these animals (FIG. 13), it is upregulated inthe RPE cell layer. It is important to note also, the expression ofOCTN1 in other retinal regions namely, the retinal ganglion cell (rgc)layer and, the filamentous labeling from the rgcs to outer nuclear layer(onl), a pattern of localization consistent with the labeling of Mullercells. These data are congruent with our previous analysis of HbFprotein expression, evaluated using immunofluorescence in similarcryosections, which revealed the MMF-induced upregulation of HbF in theRPE cell layer and throughout the neural retina (see FIG. 8).

qPCR analysis of OCTN1 mRNA expression in primary RPE, Muller andganglion cells isolated from normal mouse retinas revealed expression ofOCTN1 all three retinal cell types (FIG. 15). Interestingly, OCTN1expression appeared to be highest in pGC's. The axons of the GC'scommunicate directly with the brain for higher visualprocessing/enabling of sight as they actually bundle as they exit retinato form the optic nerve. In keeping with this, an increase in OCTN1expression and likely also HbF protein induced by MMF in these cellswould be highly beneficial in protecting these neurons from the damagingeffects of hypoxia, oxidative stress and inflammation producedcharacteristically in sickle cell disease.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs. Publications cited herein andthe materials for which they are cited are specifically incorporated byreference.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

We claim:
 1. A method of increasing fetal hemoglobin (HbF) expression ina subject in need thereof comprising administering to the subject aneffective amount of a fumaric acid ester, or pharmacologically activesalt, derivative, analogue, or prodrug thereof to increase fetalhemoglobin (HbF) expression, to induce expression of OCTN1 in theerythroid or retinal cells of the subject, or a combination thereof inthe subject.
 2. The method of claim 1, wherein the fumaric acid ester isselected from the group consisting of monomethyl fumarate (MMF),dimethyl fumarate (DMF), and a combination thereof.
 3. The method ofclaim 1, further comprising administering hydroxyurea (HU) to thesubject in combination or alternation with the fumaric acid ester orpharmacologically active salt, derivative, analogue, or prodrug thereof.4. The method of claim 3, wherein the subject does not express increasedlevels of HbF when treated with HU alone compared to when treated withthe fumaric acid ester or pharmacologically active salt, derivative,analogue, or prodrug thereof and HU in combination or alternation. 5.The method of claim 1 wherein the subject has one or more mutations inin the beta-globin gene (HBB gene) or an expression control sequencethereof.
 6. The method of claim 1 wherein the subject has sickle celldisease.
 7. The method of claim 1 wherein the subject has sickle cellanemia.
 8. The method of claim 1 wherein the subject has abeta-thalassemia.
 9. The method of claim 1 wherein the subject has asickle cell related disorder.
 10. The method of claim 1 wherein thesubject has sickle cell retinopathy.
 11. The method of claim 1 whereinthe subject has one or more symptoms of a beta-thalassemia, ahemoglobinopathy, or sickle cell disease.
 12. The method of claim 11wherein the subject has one or more symptoms of a beta-thalassemiaselected from the group consisting of anemia, fatigue and weakness, paleskin, jaundice, protruding abdomen, enlarged spleen and liver, darkurine, abnormal facial bones, poor growth, poor appetite.
 13. The methodof claim 1 wherein the subject has one or more symptoms of the sicklecell disease is selected from the group consisting of chronic hemolyticanemia, vaso-occlusive crisis, infarction of bone and bone marrow,compensatory bone marrow hyperplasia, secondary osteomyelitis, secondarygrowth defects, intravascular thrombosis, osteonecrosis, degenerativebone and joint destruction, osteolysis, articular disintegration,myelosclerosis, periosteal reaction, H-shaped vertebrae, dystrophicmedullary calcification, bone-within-bone appearance, decreased densityof the skull, decreased thickness of outer table of skull due towidening of diploe, hair on-end striations of the calvaria, osteoporosissometimes leading to biconcave vertebrae, coarsening of trabeculae inlong and flat bones, pathologic fractures, bone shortening, epiphysealdeformity with cupped metaphysis, peg-in-hole defect of distal femur,decreased height of vertebrae, hematuria, proximal tubule dysfunction,impaired potassium excretion, hyperkalemia, hypertrophied kidney, spleenenlargement, splenic sequestration crisis, infarction, low pH and lowoxygen tension in the sinusoids and splenic cords, spleen functionalimpairment, autosplenectomy, immune deficiency, increased risk ofsepsis, lower serum immunoglobulin M (IgM) levels, impairedopsonization, sluggish alternative complement pathway activation,increased susceptibility to infection pneumonia, bronchitis,cholecystitis, pyelonephritis, cystitis, osteomyelitis, meningitis,sepsis and other challenges from infectious agents, growth delays,maturation delays during puberty in adolescents, hand-foot syndrome,acute chest syndrome, stroke, hemiparesis, hemosiderin deposition in themyocardium, dilation of both ventricles and the left atrium,cholelithiasis, paraorbital facial infarction, retinal vascular changes,proliferative retinitis, loss of vision, leg ulcers, priapism, avascularnecrosis, and pulmonary hypertension.
 14. A method of increasing HbFexpression in hemoglobin producing cells comprising contacting the cellswith an effective amount of a fumaric acid ester, or pharmacologicallyactive salt, derivative, analogue, or prodrug thereof to increase HbFexpression, to induce expression of OCTN1, or a combination thereof inthe cells.
 15. The method of claim 14 further comprising contacting thecells with hydroxyurea.
 16. The method of claim 14 wherein thecontacting occurs in vitro.
 17. The method of claim 14 wherein thecontacting occurs in vivo.
 18. The method of claim 14 wherein the cellsare erythroid precursor cells, macrophage, retinal pigment cells, oralveolar epithelial cells.
 19. A method for treating sickle cell diseasecomprising administering to a subject in need thereof an effectiveamount of fumaric acid ester, or pharmacologically active salt thereofto increase fetal hemoglobin (HbF) expression in the subject.
 20. Themethod of claim 19, further comprising administering an effective amountof hydroxyurea to the subject.